Supplementary Materials for · Supplementary Materials: Materials and Methods Candidate biomarkers...

www.sciencetranslationalmedicine.org/cgi/content/full/5/207/207ra142/DC1

Supplementary Materials for

A Blood-Based Proteomic Classifier for the Molecular Characterization of Pulmonary Nodules

Xiao-jun Li,* Clive Hayward, Pui-Yee Fong, Michel Dominguez, Stephen W.

Hunsucker, Lik Wee Lee, Matthew McLean, Scott Law, Heather Butler, Michael Schirm, Olivier Gingras, Julie Lamontagne, Rene Allard, Daniel Chelsky, Nathan D. Price,

Stephen Lam, Pierre P. Massion, Harvey Pass, William N. Rom, Anil Vachani, Kenneth C. Fang, Leroy Hood, Paul Kearney*

*Corresponding author. E-mail: [email protected] (X.-j.L.); [email protected] (P.K.)

Published 16 October 2013, Sci. Transl. Med. 5, 207ra142 (2013)

DOI: 10.1126/scitranslmed.3007013

This PDF file includes:

Materials and Methods Fig. S1. Summary of 388 lung cancer biomarker candidates. Fig. S2. Summary of the 371-protein MRM assay. Fig. S3. ROC curves of the classifier in discovery and validation. Fig. S4. Scatter plot of nodule size versus classifier score of all 247 patients. Fig. S5. Performance of individual proteins as normalizers. Table S1. Clinical information of lung cancer patients enrolled in tissue biomarker studies. Table S2. Terminologies used for searching potential biomarkers in literature. Table S3. Information of 388 protein candidates. Table S4. Summary of protein MRM assay development and detection in blood. Table S6. Experimental design of the discovery study. Table S7. The list of 36 cooperative proteins. Table S8. Results from logistic regression diagnostics. Table S9. Experimental design of the validation study. Table S10. Impact of clinical characteristics on classifier score. Table S11. List of endogenous normalizing proteins. Table S12. Protein and peptide detection in the discovery study. References (50–71)

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/5/207/207ra142/DC1)

Table S5 (.pdf format). MRM assay of individual proteins.

Submitted Manuscript: Confidential

1

Supplementary Materials:

Materials and Methods

Candidate biomarkers identified by tissue proteomics. Specimens of resected

adenocarcinoma, squamous cell and large cell lung tumors and non-adjacent normal

tissue in the same lobe were obtained from patients who provided informed consent in

studies approved by the Ethics Review Boards at the Centre Hospitalier de l’Université

de Montréal and the McGill University Health Centre.

The proteomic analyses of lung tumor tissues targeted membrane-associated proteins on

endothelial cells (adenocarcinoma, n=13; squamous cell, n=18; and large cell, n=7) and

epithelial cells (adenocarcinoma, n=19; squamous cell, n=6; and large cell, n=5), and

those associated with the Golgi apparatus (adenocarcinoma, n=13; squamous cell, n=15;

and large cell, n=5): See Table S1.

Membrane proteins from endothelial cells or epithelial cells and secreted proteins were

isolated from normal or tumor tissues from fresh lung resections after washing in buffer

and disruption with a Polytron to prepare homogenates. The cell membrane protocol

included filtration using 180 µm mesh and centrifugation at 900 x g for 10 min at 4°C,

supernatants prior to layering on 50% (w:v) sucrose and centrifugation at 218,000 x g for

1 h at 4°C to pellet the membranes. Membrane pellets were resuspended and treated with

micrococcal nuclease, and incubated with the following antibodies specified by plasma

membrane type: endothelial membranes (anti-thrombomodulin, anti-ACE, anti-CD34 and

anti-CD144 antibodies); epithelial membranes (anti-ESA, anti-CEA, anti-CD66c and


2

anti-EMA antibodies), prior to centrifugation on top of a 50%(w:v) sucrose cushion at

280,000 x g (endothelial) or 218,000 x g (epithelial) for 1 h at 4°C. After pellet

resuspension, plasma membranes were isolated using MACS microbeads. Endothelial

plasma membranes were treated with KI to remove cytoplasmic peripheral proteins. The

eluate of epithelial plasma membranes was centrifuged at 337,000 x g for 30 min at 4°C

over a 33%(w:v) sucrose cushion, with resuspension of the pellet in Laemmli/Urea/DTT

after removal of the supernatant and sucrose cushion.

To isolate secreted tissue proteins, the density of the tissue homogenates (prepared as

described above) was adjusted to 1.4 M sucrose prior to isolating the secretory vesicles

by isopycnic centrifugation at 100,000 x g for 2 h at 4°C on a 0.8 and 1.2 M

discontinuous sucrose gradient. Vesicles concentrating at the 0.8/1.2 M interface were

collected and further incubated for 25 min with 0.5 M KCl to remove loosely bound

peripheral proteins. Vesicles were recuperated by ultracentrifugation at 150,000 x g for 1

h at 4°C and then opened with 100 mM (NH4)HCO3 (pH 11.0) for 30 min at 4°C.

Secreted proteins were recovered in the supernatant following ultracentrifugation at

150,000 x g for 1 h at 4°C.

Membrane or secreted proteins were then analyzed by CellCarta (Caprion) proteomics

platform, including digestion by trypsin, separation by strong cation exchange

chromatography, and analysis by reversed-phase liquid chromatography coupled with

electrospray tandem mass spectrometry (MS/MS). Peptides in the samples were

identified by database searching of MS/MS spectra using MASCOT (50) and quantified

by a label-free approach based on their signal intensity in the samples, similar to those


3

described in the literature (51-53). Proteins whose tumor-to-normal abundance ratio was

either ≥1.5 or ≤2/3 were then identified as candidate biomarkers. The results are provided

in Table S3.

Candidate biomarkers identified by literature searches. Automated literature searches

using predefined terms detailed in Table S2 and automated PERL scripts were performed

on the following databases: UniProt (www.uniprot.org ) on May 6, 2010, Entrez

(www.ncbi.nlm.nih.gov/books/NBK3836) on May 17, 2010, and NextBio

(www.nextbio.com ) on July 8, 2010. Biomarker candidates were compiled and mapped

to UniProt identifiers using the UniProt Knowledge Base

(http://www.uniprot.org/help/uniprotkb). The results are provided in Table S3.

Presence of candidate biomarkers in the blood. The tissue- and literature-identified

biomarker candidates were required to demonstrate documented evidence in the literature

or in a database as a soluble or solubilized circulating protein. The first criterion was

evidence by mass spectrometry detection, with a candidate designated as previously

detected by the following database-specific criteria: a minimum of 2 peptides in

HUPO9504, which contains 9,504 human proteins identified by MS/MS (54); a minimum

of 1 peptide in HUPO889, which is a higher confidence subset of HUPO9504 containing

889 human proteins (55); or at least 2 peptides in Peptide Atlas (56) (November 2009

build). The second criterion was annotation as either a secreted or single-pass membrane

protein in UniProt. The third criterion was designation as a plasma protein in the

literature (57). The fourth criterion was prediction as a secreted protein based on the use

of various programs: prediction by TMHMM (58) as a protein with one transmembrane


4

domain, which however is cleaved based on prediction by SignalP (59); or prediction by

TMHMM as having no transmembrane domain and prediction by either SignalP (59) or

SecretomeP (60) as a secreted protein. All candidate proteins satisfied one or more of the

criteria. The results are provided in Table S3.

Sample, subject and lung nodule inclusion and exclusion criteria. The inclusion criteria

for plasma samples were collection in EDTA-containing blood tubes; obtained from

subjects previously enrolled in the Ethics Review Board (ERB) or the Institutional

Review Boards (IRB) approved studies at the participating institutions; and archived, e.g.

labeled, aliquoted and frozen, as stipulated by the study protocols.

The inclusion criteria for subjects were the following: age ≥ 40; any smoking status, e.g.

current, former, or never; any co-morbid conditions, e.g. chronic obstructive pulmonary

disease (COPD); any prior malignancy with a minimum of 5 years in clinical remission;

any prior history of skin carcinomas, e.g. squamous or basal cell. The only exclusion

criterion was prior malignancy within 5 years of lung nodule diagnosis.

The inclusion criteria for the lung nodules included radiologic, histopathologic and

staging parameters. The radiologic criteria included size ≥ 4 mm and ≤ 30 mm, and any

spiculation or ground glass opacity. The histopathologic criteria included either diagnosis

of malignancy, e.g. non-small cell lung cancer (NSCLC), including adenocarcinoma,

bronchioloalveolar carcinoma (BAC), squamous, or large cell, or a benign process,

including inflammatory (e.g. granulomatous, infectious) ornon-inflammatory (e.g.

hamartoma) processes. The clinical staging parameters included: primary tumor: ≤T1

(e.g. 1A and 1B); regional lymph nodes: N0 or N1 only; distant metastasis: M0 only. The


5

exclusion criteria for lung nodules included the following: nodule size data unavailable;

no pathology data available, histopathologic diagnosis of small cell lung cancer; and the

following clinical staging parameters: primary tumor: ≥T2, regional lymph nodes: ≥N2,

and distant metastasis: ≥M1.

Sample layout. Up to 15 paired samples per batch were assigned randomly and iteratively

to experimental processing batches until no statistical bias was demonstrable on age,

gender or nodule size. Paired samples within each processing batch were further

randomly and repeatedly assigned to positions within the processing batch until the

absolute values of the corresponding Pearson correlation coefficients between position

and age, gender and nodule size were less than 0.1. Each pair of cancer and benign

samples was then randomized to their relative positions in the batch. To provide a

positive control for quality assessment, three 200 µl aliquots of a pooled human plasma

standard (HPS) (Bioreclamation) were positioned at the beginning, middle and end of

each processing batch, respectively. Samples within a batch were analyzed together:

sequentially during immunodepletion and MRM-MS analysis but in parallel during

denaturing, digestion, and desalting. Details of the sample layouts in the discovery and

the validation study are provided in Tables S6 and S9.

Power analysis for the classifier discovery study. The power analysis for the discovery

study was based on the following assumptions: (A) The overall false positive rate (�) was

set to 0.05. (B) Šidák correction for multiple testing (61) was used to calculate the

effective �� for testing 200 proteins, i.e. �� 1 � √1 � �� . (C) The effective

sample size was reduced by a factor of 0.864 to account for the larger sample requirement


6

for the Mann-Whitney test than for the t-test (62). (D) The overall coefficient of variation

was set to 0.43 based on a previous experience. (E) The power (1-β) of the study was

calculated based on the formula (63) for the two-sample, two-sided t-test, using effective

�� and effective sample size.

Power analysis for the classifier validation study. Sufficient cancer and benign samples

were needed in the validation study to confirm the performance of the lung nodule

classifier obtained from the discovery study. We were interested in obtaining the 95%

confidence intervals (CIs) on NPV and specificity for the classifier. Assuming the cancer

prevalence of lung nodules was �� , the negative predictive value (NPV) and the

positive predictive value (PPV) of a classifier on the intended use population with lung

nodules were calculated from sensitivity (��) and specificity (��) as follows:

�� ∗��

��∗��∗�� (S1)

�� ∗��∗��∗��

(S2)

Using Eq. (S1) above, we derived sensitivity as a function of NPV and specificity, i.e.

�� 1 � �� !" !"

��

�� (S3)

If the validation study contained �# cancer samples and �$ benign samples, variances of

sensitivity and specificity based on binomial distribution were given by (64)

%�� ∗ �1 � ��/�# (S4)


7

%�� ∗ �1 � ��/�$ (S5)

Using Eqs. (S1, S2) above, the corresponding variances of NPV and PPV were derived

under the large-sample, normal-distribution approximation as (64)

%�� '�1 � ��'(�)��

��* �)��

��+, (S6)

%�� '�1 � ��'(�)��

��* �)��

��+. (S7)

The two-sided 95% CIs of sensitivity, specificity, NPV and PPV were then given by

,-��./'/ %��, ,-��./'/ %��, ,-��./'/ %�� and

,-��./'/ %��, respectively, where -��./' � 1.959964 was the 97.5% quantile of

the normal distribution.

Immunoaffinity chromatography. An immunoaffinity column was prepared by adding 10

ml of a 50% slurry containing a 2:1 ratio of IgY14 and SuperMix resins (Sigma Aldrich),

respectively, to a glass chromatography column (Tricorn, GE Healthcare) and allowed to

settle by gravity, yielding a 5 ml volume of resin in the column. The column was capped

and placed on an HPLC system (Agilent 1100 series) for further packing with 0.15 M

(NH4)HCO3 at 2 ml/min for 20 min, with performance assessed by replicate injections of

HPS aliquots. Column performance was assessed prior to immunoaffinity separation of

each sample batch.

To isolate low abundance proteins, 60 µl of plasma were diluted in 0.15M (NH4)HCO3

(1:2 v/v) to a 180 µl final volume and filtered using a 0.2 µm AcroPrep 96-well filter


8

plate (Pall Life Sciences). Immunoaffinity separation was conducted on a IgY14-

SuperMix column connected to an HPLC system (Agilent 1100 series) using 3 buffers

(loading/washing: 0.15 M (NH4)HCO3; stripping/elution: 0.1 M glycine, pH 2.5; and

neutralization: 0.01 M Tris-HCl and 0.15 M NaCl, pH 7.4) with a cycle comprised of

load, wash, elute, neutralization and re-equilibration lasting 36 min. The unbound and

bound fractions were monitored at 280 nm and were baseline resolved after separation.

Unbound fractions (containing the low abundance proteins) were collected for

downstream processing and analysis, and lyophilized prior to enzymatic digestion.

Enzymatic digestion and solid-phase extraction. Lyophilized fractions containing low

abundance proteins were digested with trypsin after being reconstituted under mild

denaturing conditions in 200 µl of 1:1 0.1 M (NH4)HCO3 /trifluoroethanol (TFE) (v/v)

and then allowed to incubate on an orbital shaker for 30 min at RT. Samples were diluted

in 800 µl of 0.1 M (NH4)HCO3 and digested with 0.4 µg trypsin (Princeton Separations)

per sample for 16 h at 37 °C and lyophilized. Lyophilized tryptic peptides were

reconstituted in 350 µl of 0.01 M (NH4)HCO3 and incubated on an orbital shaker for 15

min at RT, followed by reduction using 30 µl of 0.05 M TCEP and incubation for 1 h at

RT and dilution in 375 µl of 90% water/10% acetonitrile/0.2% trifluoroacetic acid. The

extraction plate (Empore C18, 3M Bioanalytical Technologies) was conditioned

according to the manufacturer's protocol, and after sample loading were washed in 500 µl

of 95% water/5% acetonitrile/0.1% trifluroacetic acid and eluted by 200 µl of 52%

water/48% acetonitrile/0.1% trifluoroacetic acid into a collection plate. The eluate was

split into 2 equal aliquots and was taken to dryness in a vacuum concentrator. One aliquot

was used immediately for mass spectrometry, while the other was stored at -80°C.


9

Samples were reconstituted in 12 µl of 90% water/10% acetonitrile/0.2% formic acid just

prior to LC-MRM MS analysis.

Development of MRM assays. MRM assays for candidate proteins were developed based

on synthetic peptides (17, 26, 33). After identification and synthesis of up to five suitable

peptides per protein, MRM triggered MS/MS spectra were collected on a 5500 QTrap

mass spectrometer for both doubly and triply charged precursor ions. The obtained

MS/MS spectra were assigned to peptides using MASCOT (50). Since the MS/MS

spectra were collected from the mixture of synthetic peptides, a minimum cutoff score of

15 was used in peptide assignment. Up to four transitions per precursor ion were then

selected for optimization. The resulting corresponding optimal retention time,

declustering potential and collision energy were assembled for all transitions. Optimal

transitions were measured on a mixture of all synthetic peptides and on two pooled

plasma samples, each obtained from ten subjects with either benign or malignant, i.e.

NSCLC, lung nodules at the Institut Universitaire de Cardiologie et de Pneumologie de

Quebec (IUCPQ). All subjects provided informed consent and contributed biospecimens

in studies approved by the institution’s Ethics Review Board (ERB). Plasma samples

were processed as described above. Batches of 1750 transitions were analyzed by MRM-

MS, as described below. The two best transition per peptide and two best peptides per

protein were selected manually. The intensity ratio between the two best transitions of a

peptide in the synthetic peptide mixture, was used to assess the specificity of the

transitions in a biological sample. Transitions demonstrating interference with other

transitions were not selected. A method of calculating q values of peptide and protein

assays was developed to ensure that the observed transitions corresponded to the peptides


10

and proteins they were intended to measure, as described below. About 93% of peptide

transitions developed had an error rate below 5%.

MRM-MS analysis. Peptide samples were separated using a capillary reversed-phase LC

column (Thermo BioBasic 18 KAPPA; column dimensions: 320 µm x 150 mm; particle

size: 5 µm; pore size: 300 Å) and a nano-HPLC system (nanoACQUITY, Waters Inc.).

The mobile phases were (A) 0.2% formic acid in water and (B) 0.2% formic acid in

acetonitrile. The samples were injected (8 µl) and separated using a linear gradient (98%

A to 70% A) at 5 µl/minute for 19 min. Peptides were eluted directly into the electrospray

source of the mass spectrometer (5500 QTrap LC/MS/MS, AB Sciex) operating in

scheduled MRM positive-ion mode (Q1 resolution: unit; Q3 resolution: unit; detection

window: 180 seconds; cycle time: 1.5 seconds). Transition intensities were then

integrated by software MultiQuant (AB Sciex). An intensity threshold of 10,000 was used

to filter out non-specific data and undetected transitions.

Calculation of q-values of peptide and protein assays. In the development of MRM

assays, it is important to ensure that the transitions detected correspond to the peptides

and proteins they were intended to measure. Computational tools such as mProphet (65)

enable automated qualification of MRM assays. We introduced a complementary strategy

to mProphet that does not require customization for each dataset. It utilizes expression

correlation techniques (66) to confirm the identity of transitions from the same peptide

and protein with high confidence. In Fig. S2(B), a histogram of the Pearson correlations

between every pair of transitions in the assay is presented. The correlation between a pair

of transitions was obtained from their expression profiles over all samples in the


11

discovery study. As expected, transitions from the same peptide were highly correlated.

Similarly, transitions from different peptide fragments of the same protein were also

highly correlated. In contrast, transitions from different proteins were not highly

correlated, which enabled a statistical analysis of the quality of a protein’s MRM assay.

To determine the false positive assay rate we calculated the q-values (67) of peptide

MRM assays. Using the distribution of Pearson correlations between transitions

from different proteins as the null distribution (Fig. S2(B)), an empirical p-value was

assigned to a pair of transitions from the same peptide, detected in at least five

common samples. A value of ‘NA’ was assigned if the pair of transitions were

detected in less than five common samples. The empirical p-value was converted to

a q-value using the “qvalue” package in Bioconductor

(www.bioconductor.org/packages/release/bioc/html/qvalue.html). We calculated

the q-values of protein MRM assays in the same way except Pearson correlations of

individual proteins were calculated as those between two transitions from different

peptides of the protein. For proteins not having two peptides detected in five or

more common samples, their q-values could not be properly evaluated and were

assigned ‘NA’. If the correlation of transitions from two peptides from the same

protein was above 0.5 then there was less than a 3% probability that the assay was

false. See Table S12 for more details.

Definition of depletion column drift. Due to changes in observed signal intensity after

repetitive use of each immunoaffinity column, the column's performance was assessed by


12

quantifying the transition intensity in the control HPS samples. Assuming 56,� was the

intensity of transition 8 in an HPS sample �, the drift of the sample was defined as

9�8:;� � <�98%��=>,?�=@?=@?�, (S8)

where 5@6 was the mean value of 56,� among all HPS samples that were depleted by the

same column, and the median was taken over all detected transitions in the sample. The

column variability, or drift, was defined as

9�8:;�AB � <�98%��9�8:;� C 0� � <�98%��9�8:;� E 0�. (S9)

Here the median was taken over all HPS samples depleted by the column. If no sample

drift were greater or less than zero, the corresponding median was taken as 0. The median

column drift was the median of drifts of all depletion columns used in the study.

Calibration by human plasma standard (HPS) samples. For a label-free MS approach,

variation on signal intensity between different experiments is expected. To reduce this

variation, we utilized HPS samples as an external standard and calibrated the intensity

between the discovery and validation studies. Assuming that 5F6,� was the logarithmically

transformed (base 2), normalized intensity of transition 8 in sample �, 5F6,G6� and 5F6,�)B were

the corresponding median values of HPS samples in the discovery and the validation

studies, respectively, then the HPS corrected intensity was

5H6,� � 5I6,� � 5I6,�)B * 5I6,G6� (S10)


13

Figures

Fig. S1. Summary of 388 lung cancer biomarker candidates. (A) Venn diagram

of sources of tissue biomarkers (see Table S1). (B) Venn diagram of biomarkers

in literature (see Table S2). (C) Overlap between tissue biomarkers and

biomarkers in literature. (D) Venn diagram of evidences for protein presence in

blood (see Table S3 for details).

7"

87"

32"

66"

4"

32"91"

Lung"cancers"(276)"

Diseases"causing"benign"lung"nodules"

(168)"

Symptoms"of"benign"or"cancerous"lung"nodules"(159)"

Total"number:"319"

Tissue"biomarkers"

(217)"

Biomarkers"in"literature"

(319)"69" 148" 171"

Total"number:"388"

UniProt"(220)"

Total"number:"388"

Literature"(173)"

40"22"42"

40"

13"

18"

10"

22"

34"

24"

10"

30"

16"12"

55"

DetecIon"(214)" PredicIon"(275)"

A B

C D

70"

24"

47"

24"

24"

8"20"

Secreted"(76)"

Cell"surface"epithelial"(138)"

Cell"surface"endothelial"

(99)"

Total"number:"217"


14

Fig. S2. Summary of the 371-protein MRM assay. (A) Protein (blue) and peptide

(green) MRM assay development and detection in blood. (B) Histograms of the

Pearson correlations for transitions from the same peptide (blue), from the same

protein (green) and from different proteins (red).

Selected'for'development'

Synthesized'pep4des'

Developed'SRM'assays'

Detected'in'blood'

Protein' 388' 381' 371' 190'

Pep4de' 1730' 1425' 344'

0'

400'

800'

1200'

1600'

2000' Protein' Pep4de'

0'

0.05'

0.1'

0.15'

0.2'

0.25'

F1' F0.8' F0.6' F0.4' F0.2' 0' 0.2' 0.4' 0.6' 0.8' 1'

freq

uency'

Pearson'correla4on'

Same'pep4de' Same'protein' Different'proteins'

A

B


15

Fig. S3. Receiver operating characteristic curves of the classifier in discovery

and validation. The corresponding areas under the receiver operating

characteristic curves (AUCs) were 0.82 and 0.60, respectively.

0.1

.2.3

.4.5

.6.7

.8.9

1Se

nsiti

vity

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 11−Specificity

Discovery Validation


16

Fig. S4. Scatter plot of nodule size vs. classifier score of all 247 patients. The

plot demonstrated the lack of correlation between the two variables. (A) Samples

in discovery and (B) samples in validation.

Classifier score

Nod

ule

size

(mm

)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0

5

10

15

20

25

30

35

40

45 BenignCancer

A

Classifier score

Nod

ule

size

(mm

)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0

5

10

15

20

25

30 BenignCancer

B


17

Fig. S5. Performance of individual proteins as normalizers. APOA1 was not

selected as a normalizer due to high biological coefficient of variation (CV).

K1C19 was not slected since it is a common contamination in mass spectrometry

analysis.

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

0 5 10 15 20

05

1015

20

Rank in reducing technical CV

Ran

k in

redu

cing

col

umn

drift

LG3BP

PEDF

APOA1

MASP1

C163A

LUM

GELS

IBP2

AMPN

PTPRJ

K1C19

ICAM3

IBP3

DSG2

APOE

CD14

COIA1

GGH●

●

●

●

●

●


18

Tables

Table S1. Clinical information of lung cancer patients enrolled in tissue biomarker studies.

Isolation Histologic type Histologic grade

Extent of invasion

Regional lymph nodes

Distant metastasis

Gender

Cell Surface (Endothelial)

adenocarcinoma G1 T2 N0 M0 F


adenocarcinoma G2 T1 N0 MX M






adenocarcinoma G2 T2 N1 MX F




adenocarcinoma G2 T3 N0 M0 M














large cell G3 T2 N0 M0 M


large cell G3 T2 N0 MX F




large cell G4 T2 N0 MX M








19



Extent of invasion


Distant metastasis

Gender


squamous G1 T2 n/a M0 M


squamous G2 T2 N0 M0 F


squamous G2 T2 N0 M0 M




squamous G2 T2 N0 MX F


squamous G2 T2 N0 MX M






squamous G2 T2 NX MX M



















Cell Surface (Epithelial)







20



Extent of invasion


Distant metastasis

Gender














adenocarcinoma G2 T2 NX MX M




adenocarcinoma G2/3 T1 N0 M0 M


adenocarcinoma G2/3 T1 N1 MX M












adenocarcinoma G3 T2 N0 MX n/a




large cell G3 T2 N0 M0 F








21



Extent of invasion


Distant metastasis

Gender




squamous G2 T2 N0 M0 F









Secreted adenocarcinoma G1 T2 n/a MX F Secreted adenocarcinoma G1 T2 N0 M0 F Secreted adenocarcinoma G2 T1 N0 MX F Secreted adenocarcinoma G2 T1 N0 MX M Secreted adenocarcinoma G2 T1 N1 MX F Secreted adenocarcinoma G2 T2 N0 M1 F Secreted adenocarcinoma G2 T2 N2 MX M Secreted adenocarcinoma G2 T3 N0 M0 M Secreted adenocarcinoma G2/3 T1 N0 M0 M Secreted adenocarcinoma G3 T1 N1 MX F Secreted adenocarcinoma G3 T1 N2 MX F Secreted adenocarcinoma G3 T2 N0 MX F Secreted adenocarcinoma G3 T2 N0 MX M Secreted large cell G3 T2 N0 M0 M Secreted large cell G3 T2 N0 MX F Secreted large cell G3 T3 N1 MX F Secreted large cell G4 T2 N0 MX M Secreted large cell G4 T2 N2 MX M Secreted squamous G1 T1 N0 MX F Secreted squamous G1 T2 N1 M0 M Secreted squamous G1 T2 NX M0 M Secreted squamous G2 T1 N1 M0 F Secreted squamous G2 T2 N0 M0 F Secreted squamous G2 T2 N0 M0 M Secreted squamous G2 T2 N0 MX M Secreted squamous G2 T2 N0 MX M Secreted squamous G3 T1 N0 MX F Secreted squamous G3 T2 N0 MX M Secreted squamous G3 T2 N1 MX M Secreted squamous G3 T2 N1 MX M Secreted squamous G3 T2 N1 MX M


22



Extent of invasion


Distant metastasis

Gender

Secreted squamous G3 T3 N0 MX M Secreted squamous G3 T3 N1 M0 M

Note:

• Histologic grade o G1 = Well differentiated. o G2 = Moderately differentiated. o G3 = Poorly differentiated. o G4 = Undifferentiated.

• Extent of invasion o T1 = Tumor ≤ 3 cm in greatest dimension, surrounded by lung or visceral

pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus.

o T2 = Tumor with any of the following features of size or extent: > 3 cm in greatest dimension, involves main bronchus, ≥ 2 cm distal to the carina, invades the visceral pleura, associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung.

o T3=Tumor of any size that directly invades any of the following Chest wall (including superior sulcus tumors), diaphragm, mediastinial pleura or parietal pericardium.

o T4 = Tumor of any size that invades any of the following: mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina.

• Regional lymph nodes o NX = Cannot be assessed. N0 = No regional lymph node metastasis. o N1 = Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph

nodes, including intrapulmonary nodes involved by direct extension of the primary tumor.

o N2 = Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s). o n/a = not available.

• Distant metastasis o MX = Cannot be assessed. o M0 = No distant metastasis. o M1 = Distant metastasis, includes separate tumor nodule(s) in a different lobe

(ipsilateral or contralateral). • Gender

o F = Female o M = Male


23

Table S2. Terminologies used for searching potential biomarkers in literature.

Lung cancer Disease causing benign

lung nodules

Symptom of cancerous

or benign lung nodules

Bronchial adenoma

Bronchioalveolar

carcinoma

Bronchioloalveolar

carcinoma

Bronchogenic carcinoma

Large cell lung cancer

Large cell lung carcinoma

Lung adenocarcinoma

Lung cancer

Lung carcinoid

Lung carcinoma

Lung carcinosarcoma

Lung chondrosarcoma

Lung lymphoma

Lung mesothelioma

Lung pleomorphic

carcinoma

Lung rhabdomysarcoma

Lung sarcoma

Lung solitary fibrous

tumor

Nonsmall cell lung cancer

Non-small cell lung

cancer

Nonsquamous cell lung

carcinoma

Non-small cell lung

cancer

Nonsquamous cell lung

carcinoma

Non-squamous cell lung

carcinoma

NSCLC

Pleura solitary fibrous

tumor

Pulmonary

adenocarcinoma

Anthracosilicosis

Asbestosis

Atelectasis

Bronchiectasis

Bronchitis

Bronchogenic cyst

Caplan's disease

Caplan's syndrome

Castleman's disease

Chronic obstructive

pulmonary disease

Cystic fibrosis

Emphysema

Eosinophilic granuloma

Fibrosing alveolitis

Hemangioma

Lung abscess

Lung actinomycosis

Lung amyloidosis

Lung arteriovenous

malformation

Lung aspergilloma

Lung aspergillosis

Lung blastoma

Lung blastomycosis

Lung coccidioidomycosis

Lung consolidation

Lung cryptococcosis

Lung cyst

Lung dirofilariasis

Lung embolism

Lung fibroma

Lung fibrosis

Lung granuloma

Lung hamartoma

Lung histoplasmosis

Lung hyalinizing

Alkaline phosphatase

Anemia

Hemoptysis

Liver enzyme

Serum calcium


24



lung nodules



Pulmonary cancer

Pulmonary carcinoid

Pulmonary carcinoma

Pulmonary

carcinosarcoma

Pulmonary

chondrosarcoma

Pulmonary lymphoma

Pulmonary

mesothelioma

Pulmonary pleomorphic

carcinoma

Pulmonary

rhabdomysarcoma

Pulmonary sarcoma

Pulmonary solitary

fibrous tumor

SCLC

Small cell lung cancer

Squamous cell lung

carcinoma

granuloma

Lung infarct

Lung infarction

Lung infection

Lung mycosis

Lung neurofibroma

Lung nocardiosis

Lung rheumatoid arthritis

Lung Sarcoidosis

Lung sequestration

Lung toxocariasis

Lung veno-occlusive

disease

Lung Wegener

granulomatosis

Lymph node hyperplasia

Pneumocystosis

Pneumonia

Pulmonary abscess

Pulmonary actinomycosis

Pulmonary amyloidosis

Pulmonary arteriovenous

malformation

Pulmonary aspergilloma

Pulmonary aspergillosis

Pulmonary blastoma

Pulmonary blastomycosis

Pulmonary

coccidioidomycosis

Pulmonary consolidation

Pulmonary

cryptococcosis

Pulmonary cyst

Pulmonary dirofilariasis

Pulmonary embolism

Pulmonary fibroma

Pulmonary fibrosis

Pulmonary granuloma

Pulmonary hamartoma

Pulmonary


25



lung nodules



histoplasmosis

Pulmonary hyalinizing

granuloma

Pulmonary infarct

Pulmonary infarction

Pulmonary infection

Pulmonary mycosis

Pulmonary neurofibroma

Pulmonary nocardiosis

Pulmonary rheumatoid

arthritis

Pulmonary Sarcoidosis

Pulmonary sequestration

Pulmonary toxocariasis

Pulmonary veno-

occlusive disease

Pulmonary Wegener

granulomatosis

Rheumatoid lung disease

Tuberculosis


26

Table S3. Information of 388 protein candidates. Protein (HUMAN)

Protein name Gene symbol Source of tissue biomarkers

Biomarkers in literature

Subcellular location (UniProt) Evidence for presence in blood

1433B 14-3-3 protein beta/alpha YWHAB Secreted, EPI LungCancers Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.

Literature, Detection

1433E 14-3-3 protein epsilon YWHAE ENDO LungCancers, BenignNodules

Cytoplasm (By similarity). Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


1433S 14-3-3 protein sigma SFN Secreted, EPI LungCancers Cytoplasm. Nucleus (By similarity). Secreted. Note=May be secreted by a non-classical secretory pathway.

UniProt, Literature, Detection

1433T 14-3-3 protein theta YWHAQ EPI LungCancers, BenignNodules

Cytoplasm. Note=In neurons, axonally transported to the nerve terminals.

Detection

1433Z 14-3-3 protein zeta/delta YWHAZ EPI LungCancers, BenignNodules

Cytoplasm. Melanosome. Note=Located to stage I to stage IV melanosomes.

Detection

6PGD 6-phosphogluconate dehydrogenase, decarboxylating

PGD EPI, ENDO Cytoplasm (By similarity). Detection

A1AG1 Alpha-1-acid glycoprotein 1 ORM1 EPI Symptoms Secreted. UniProt, Literature, Detection, Prediction

ABCD1 ATP-binding cassette sub-family D member 1

ABCD1 ENDO Peroxisome membrane; Multi-pass membrane protein.

Detection, Prediction

ADA12 Disintegrin and metalloproteinase domain-containing protein 12

ADAM12 LungCancers, BenignNodules, Symptoms

Isoform 1: Cell membrane; Single-pass type I membrane protein.|Isoform 2: Secreted.|Isoform 3: Secreted (Potential).|Isoform 4: Secreted (Potential).

UniProt, Detection, Prediction

ADML ADM ADM LungCancers, BenignNodules, Symptoms

Secreted. UniProt, Literature, Detection, Prediction


27





AGR2 Anterior gradient protein 2 homolog

AGR2 EPI LungCancers Secreted. Endoplasmic reticulum (By similarity).

UniProt, Prediction

AIFM1 Apoptosis-inducing factor 1, mitochondrial

AIFM1 EPI, ENDO LungCancers Mitochondrion intermembrane space. Nucleus. Note=Translocated to the nucleus upon induction of apoptosis.


ALDOA Fructose-bisphosphate aldolase A

ALDOA Secreted, EPI LungCancers, Symptoms


AMPN Aminopeptidase N ANPEP EPI, ENDO LungCancers, BenignNodules, Symptoms

Cell membrane; Single-pass type II membrane protein. Cytoplasm, cytosol (Potential). Note=A soluble form has also been detected.

UniProt, Detection

ANGP1 Angiopoietin-1 ANGPT1 LungCancers, BenignNodules

Secreted. UniProt, Literature, Prediction

ANGP2 Angiopoietin-2 ANGPT2 LungCancers, BenignNodules


APOA1 Apolipoprotein A-I APOA1 LungCancers, BenignNodules, Symptoms


APOE Apolipoprotein E APOE EPI, ENDO LungCancers, BenignNodules, Symptoms


ASM3B Acid sphingomyelinase-like phosphodiesterase 3b

SMPDL3B EPI, ENDO Secreted (By similarity). UniProt, Prediction

AT2A2 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2

ATP2A2 EPI, ENDO LungCancers, BenignNodules

Endoplasmic reticulum membrane; Multi-pass membrane protein. Sarcoplasmic reticulum membrane; Multi-pass membrane protein.

Detection


28





ATS1 A disintegrin and metalloproteinase with thrombospondin motifs 1

ADAMTS1 LungCancers, BenignNodules, Symptoms

Secreted, extracellular space, extracellular matrix (By similarity).

UniProt, Literature, Prediction


ADAMTS12 LungCancers Secreted, extracellular space, extracellular matrix (By similarity).



ADAMTS19 LungCancers Secreted, extracellular space, extracellular matrix (By similarity).

UniProt, Prediction

BAGE1 B melanoma antigen 1 BAGE LungCancers Secreted (Potential). UniProt, Prediction

BAGE2 B melanoma antigen 2 BAGE2 LungCancers Secreted (Potential). UniProt, Prediction




BASP1 Brain acid soluble protein 1 BASP1 Secreted, EPI Cell membrane; Lipid-anchor. Cell projection, growth cone. Note=Associated with the membranes of growth cones that form the tips of elongating axons.

Detection

BAX Apoptosis regulator BAX BAX EPI LungCancers, BenignNodules

Isoform Alpha: Mitochondrion membrane; Single-pass membrane protein. Cytoplasm. Note=Colocalizes with 14- 3-3 proteins in the cytoplasm. Under stress conditions, redistributes to the mitochondrion membrane through the release from JNK-phosphorylated 14-3-3 proteins.|Isoform Beta: Cytoplasm.|Isoform Gamma: Cytoplasm.|Isoform Delta: Cytoplasm (Potential).



29





BDNF Brain-derived neurotrophic factor

BDNF BenignNodules, Symptoms


BGH3 Transforming growth factor-beta-induced protein ig-h3

TGFBI LungCancers, BenignNodules

Secreted, extracellular space, extracellular matrix. Note=May be associated both with microfibrils and with the cell surface.

UniProt, Detection

BMP2 Bone morphogenetic protein 2 BMP2 LungCancers, BenignNodules, Symptoms

Secreted. UniProt, Literature

BST1 ADP-ribosyl cyclase 2 BST1 EPI Symptoms Cell membrane; Lipid-anchor, GPI-anchor. Detection, Prediction

C163A Scavenger receptor cysteine-rich type 1 protein M130

CD163 EPI Symptoms Soluble CD163: Secreted.|Cell membrane; Single-pass type I membrane protein. Note=Isoform 1 and isoform 2 show a lower surface expression when expressed in cells.

UniProt, Detection

C4BPA C4b-binding protein alpha chain

C4BPA LungCancers, Symptoms

Secreted. UniProt, Detection, Prediction

CAH9 Carbonic anhydrase 9 CA9 LungCancers, BenignNodules, Symptoms

Nucleus. Nucleus, nucleolus. Cell membrane; Single-pass type I membrane protein. Cell projection, microvillus membrane; Single-pass type I membrane protein. Note=Found on the surface microvilli and in the nucleus, particularly in nucleolus.

UniProt

CALR Calreticulin CALR EPI Symptoms Endoplasmic reticulum lumen. Cytoplasm, cytosol. Secreted, extracellular space, extracellular matrix. Cell surface. Note=Also found in cell surface (T cells), cytosol and extracellular matrix. Associated with the lytic granules in the cytolytic T-lymphocytes.

UniProt, Literature, Detection, Prediction


30





CALU Calumenin CALU EPI Symptoms Endoplasmic reticulum lumen. Secreted. Melanosome. Sarcoplasmic reticulum lumen (By similarity). Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


CALX Calnexin CANX Secreted, EPI, ENDO

BenignNodules Endoplasmic reticulum membrane; Single-pass type I membrane protein. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


CAP7 Azurocidin AZU1 EPI Symptoms Cytoplasmic granule. Note=Cytoplasmic granules of neutrophils.

Prediction

CATB Cathepsin B CTSB Secreted LungCancers Lysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.

Literature, Detection, Prediction

CATG Cathepsin G CTSG Secreted, ENDO

BenignNodules Cell surface. Detection, Prediction

CBPB2 Carboxypeptidase B2 CPB2 LungCancers, BenignNodules, Symptoms


CCL22 C-C motif chemokine 22 CCL22 LungCancers, BenignNodules

Secreted. UniProt, Prediction

CD14 Monocyte differentiation antigen CD14

CD14 EPI LungCancers, BenignNodules, Symptoms

Cell membrane; Lipid-anchor, GPI-anchor. Literature, Detection, Prediction

CD24 Signal transducer CD24 CD24 LungCancers, BenignNodules

Cell membrane; Lipid-anchor, GPI-anchor. Literature

CD2A2 Cyclin-dependent kinase inhibitor 2A, isoform 4

CDKN2A LungCancers, BenignNodules

Cytoplasm. Nucleus.|Nucleus, nucleolus (By similarity).

Literature, Prediction

CD38 ADP-ribosyl cyclase 1 CD38 EPI, ENDO Symptoms Membrane; Single-pass type II membrane protein.

UniProt, Literature


31





CD40L CD40 ligand CD40LG LungCancers, BenignNodules, Symptoms

Cell membrane; Single-pass type II membrane protein.|CD40 ligand, soluble form: Secreted.

UniProt, Literature

CD44 CD44 antigen CD44 EPI LungCancers, BenignNodules, Symptoms

Membrane; Single-pass type I membrane protein.


CD59 CD59 glycoprotein CD59 LungCancers, BenignNodules, Symptoms

Cell membrane; Lipid-anchor, GPI-anchor. Secreted. Note=Soluble form found in a number of tissues.


CD97 CD97 antigen CD97 EPI, ENDO Symptoms Cell membrane; Multi-pass membrane protein.|CD97 antigen subunit alpha: Secreted, extracellular space.

UniProt

CDCP1 CUB domain-containing protein 1

CDCP1 LungCancers Isoform 1: Cell membrane; Single-pass membrane protein (Potential). Note=Shedding may also lead to a soluble peptide.|Isoform 3: Secreted.

UniProt, Prediction

CDK4 Cell division protein kinase 4 CDK4 LungCancers, Symptoms

Literature

CEAM5 Carcinoembryonic antigen-related cell adhesion molecule 5

CEACAM5 EPI LungCancers, BenignNodules, Symptoms

Cell membrane; Lipid-anchor, GPI-anchor. Literature, Prediction

CEAM8 Carcinoembryonic antigen-related cell adhesion molecule 8

CEACAM8 EPI LungCancers Cell membrane; Lipid-anchor, GPI-anchor. Detection, Prediction

CERU Ceruloplasmin CP EPI LungCancers, Symptoms


CH10 10 kDa heat shock protein, mitochondrial

HSPE1 ENDO LungCancers Mitochondrion matrix. Literature, Detection, Prediction


32





CH60 60 kDa heat shock protein, mitochondrial

HSPD1 Secreted, EPI, ENDO

LungCancers, Symptoms

Mitochondrion matrix. Literature, Detection

CKAP4 Cytoskeleton-associated protein 4

CKAP4 EPI, ENDO LungCancers Endoplasmic reticulum-Golgi intermediate compartment membrane; Single-pass membrane protein (Potential).

UniProt

CL041 Uncharacterized protein C12orf41

C12orf41 ENDO Prediction

CLCA1 Calcium-activated chloride channel regulator 1

CLCA1 LungCancers, BenignNodules

Secreted, extracellular space. Cell membrane; Peripheral membrane protein; Extracellular side. Note=Protein that remains attached to the plasma membrane appeared to be predominantly localized to microvilli.

UniProt, Prediction

CLIC1 Chloride intracellular channel protein 1

CLIC1 EPI Nucleus. Nucleus membrane; Single-pass membrane protein (Probable). Cytoplasm. Cell membrane; Single-pass membrane protein (Probable). Note=Mostly in the nucleus including in the nuclear membrane. Small amount in the cytoplasm and the plasma membrane. Exists both as soluble cytoplasmic protein and as membrane protein with probably a single transmembrane domain.


CLUS Clusterin CLU EPI, ENDO LungCancers, BenignNodules, Symptoms


CMGA Chromogranin-A CHGA LungCancers, BenignNodules

Secreted. Note=Neuroendocrine and endocrine secretory granules.



33





CNTN1 Contactin-1 CNTN1 LungCancers Isoform 1: Cell membrane; Lipid-anchor, GPI- anchor; Extracellular side.|Isoform 2: Cell membrane; Lipid-anchor, GPI- anchor; Extracellular side.


CO4A1 Collagen alpha-1(IV) chain COL4A1 LungCancers Secreted, extracellular space, extracellular matrix, basement membrane.


CO5A2 Collagen alpha-2(V) chain COL5A2 LungCancers Secreted, extracellular space, extracellular matrix (By similarity).


CO6A3 Collagen alpha-3(VI) chain COL6A3 Secreted Symptoms Secreted, extracellular space, extracellular matrix (By similarity).


COCA1 Collagen alpha-1(XII) chain COL12A1 ENDO LungCancers, Symptoms


UniProt, Prediction

COF1 Cofilin-1 CFL1 Secreted, EPI LungCancers, BenignNodules

Nucleus matrix. Cytoplasm, cytoskeleton. Note=Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide.


COIA1 Collagen alpha-1(XVIII) chain COL18A1 LungCancers, BenignNodules



COX5A Cytochrome c oxidase subunit 5A, mitochondrial

COX5A Secreted, ENDO

Mitochondrion inner membrane. Prediction

CRP C-reactive protein CRP LungCancers, BenignNodules, Symptoms


CS051 UPF0470 protein C19orf51 C19orf51 ENDO Prediction


34





CSF1 Macrophage colony-stimulating factor 1

CSF1 LungCancers, BenignNodules

Cell membrane; Single-pass membrane protein (By similarity).|Processed macrophage colony-stimulating factor 1: Secreted, extracellular space (By similarity).


CSF2 Granulocyte-macrophage colony-stimulating factor

CSF2 LungCancers, BenignNodules


CT085 Uncharacterized protein C20orf85

C20orf85 LungCancers, BenignNodules

Prediction

CTGF Connective tissue growth factor CTGF LungCancers, BenignNodules

Secreted, extracellular space, extracellular matrix (By similarity). Secreted (By similarity).


CYR61 Protein CYR61 CYR61 LungCancers, BenignNodules


CYTA Cystatin-A CSTA LungCancers Cytoplasm. Literature, Detection

CYTB Cystatin-B CSTB Secreted Cytoplasm. Nucleus. Literature, Detection

DDX17 Probable ATP-dependent RNA helicase DDX17

DDX17 ENDO LungCancers, BenignNodules

Nucleus. Detection, Prediction

DEFB1 Beta-defensin 1 DEFB1 LungCancers, BenignNodules


DESP Desmoplakin DSP EPI, ENDO LungCancers Cell junction, desmosome. Cytoplasm, cytoskeleton. Note=Innermost portion of the desmosomal plaque.

Detection

DFB4A Beta-defensin 4A DEFB4A LungCancers, BenignNodules

Secreted. UniProt

DHI1L Hydroxysteroid 11-beta-dehydrogenase 1-like protein

HSD11B1L LungCancers Secreted (Potential). UniProt, Prediction


35





DMBT1 Deleted in malignant brain tumors 1 protein

DMBT1 LungCancers, BenignNodules

Secreted (By similarity). Note=Some isoforms may be membrane-bound. Localized to the lumenal aspect of crypt cells in the small intestine. In the colon, seen in the lumenal aspect of surface epithelial cells. Formed in the ducts of von Ebner gland, and released into the fluid bathing the taste buds contained in the taste papillae (By similarity).


DMKN Dermokine DMKN LungCancers Secreted. UniProt, Detection, Prediction

DPP4 Dipeptidyl peptidase 4 DPP4 EPI LungCancers, BenignNodules, Symptoms

Dipeptidyl peptidase 4 soluble form: Secreted.|Cell membrane; Single-pass type II membrane protein.

UniProt, Detection

DSG2 Desmoglein-2 DSG2 ENDO Symptoms Cell membrane; Single-pass type I membrane protein. Cell junction, desmosome.

UniProt, Detection

DX39A ATP-dependent RNA helicase DDX39A

DDX39A EPI Nucleus (By similarity). Prediction

DX39B Spliceosome RNA helicase DDX39B

DDX39B EPI Nucleus. Nucleus speckle. Prediction

DYRK2 Dual specificity tyrosine-phosphorylation-regulated kinase 2

DYRK2 ENDO LungCancers Cytoplasm. Nucleus. Note=Translocates into the nucleus following DNA damage.

Literature

EDN2 Endothelin-2 EDN2 LungCancers Secreted. UniProt, Prediction

EF1A1 Elongation factor 1-alpha 1 EEF1A1 Secreted, EPI LungCancers, BenignNodules

Cytoplasm. Detection

EF1D Elongation factor 1-delta EEF1D Secreted, EPI LungCancers Prediction EF2 Elongation factor 2 EEF2 Secreted, EPI Cytoplasm. Literature,

Detection


36





EGF Pro-epidermal growth factor EGF LungCancers, BenignNodules, Symptoms


UniProt, Literature

EGFL6 Epidermal growth factor-like protein 6

EGFL6 LungCancers Secreted, extracellular space, extracellular matrix, basement membrane (By similarity).


ENOA Alpha-enolase ENO1 Secreted, EPI, ENDO

LungCancers, BenignNodules, Symptoms

Cytoplasm. Cell membrane. Cytoplasm, myofibril, sarcomere, M-band. Note=Can translocate to the plasma membrane in either the homodimeric (alpha/alpha) or heterodimeric (alpha/gamma) form. ENO1 is localized to the M-band.|Isoform MBP-1: Nucleus.


ENOG Gamma-enolase ENO2 EPI LungCancers, Symptoms

Cytoplasm (By similarity). Cell membrane (By similarity). Note=Can translocate to the plasma membrane in either the homodimeric (alpha/alpha) or heterodimeric (alpha/gamma) form (By similarity).


ENOX2 Ecto-NOX disulfide-thiol exchanger 2

ENOX2 LungCancers Cell membrane. Secreted, extracellular space. Note=Extracellular and plasma membrane-associated.

UniProt, Detection

ENPL Endoplasmin HSP90B1 Secreted, EPI, ENDO


Endoplasmic reticulum lumen. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


EPHB6 Ephrin type-B receptor 6 EPHB6 LungCancers Membrane; Single-pass type I membrane protein.|Isoform 3: Secreted (Probable).

UniProt, Literature

EPOR Erythropoietin receptor EPOR LungCancers, BenignNodules, Symptoms

Cell membrane; Single-pass type I membrane protein.|Isoform EPOR-S: Secreted. Note=Secreted and located to the cell surface.



37





ERBB3 Receptor tyrosine-protein kinase erbB-3

ERBB3 LungCancers, BenignNodules

Isoform 1: Cell membrane; Single-pass type I membrane protein.|Isoform 2: Secreted.


EREG Proepiregulin EREG LungCancers Epiregulin: Secreted, extracellular space.|Proepiregulin: Cell membrane; Single-pass type I membrane protein.

UniProt

ERO1A ERO1-like protein alpha ERO1L Secreted, EPI, ENDO

Symptoms Endoplasmic reticulum membrane; Peripheral membrane protein; Lumenal side. Note=The association with ERP44 is essential for its retention in the endoplasmic reticulum.

Prediction

ESM1 Endothelial cell-specific molecule 1

ESM1 LungCancers, BenignNodules


EZRI Ezrin EZR Secreted LungCancers, BenignNodules

Apical cell membrane; Peripheral membrane protein; Cytoplasmic side. Cell projection. Cell projection, microvillus membrane; Peripheral membrane protein; Cytoplasmic side. Cell projection, ruffle membrane; Peripheral membrane protein; Cytoplasmic side. Cytoplasm, cell cortex. Cytoplasm, cytoskeleton. Note=Localization to the apical membrane of parietal cells depends on the interaction with MPP5. Localizes to cell extensions and peripheral processes of astrocytes (By similarity). Microvillar peripheral membrane protein (cytoplasmic side).


F10A1 Hsc70-interacting protein ST13 EPI Cytoplasm (By similarity).|Cytoplasm (Probable).


FAM3C Protein FAM3C FAM3C EPI, ENDO Secreted (Potential). UniProt, Detection


38





FAS Fatty acid synthase FASN EPI LungCancers, BenignNodules, Symptoms

Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


FCGR1 High affinity immunoglobulin gamma Fc receptor I

FCGR1A EPI LungCancers, BenignNodules, Symptoms

Cell membrane; Single-pass type I membrane protein. Note=Stabilized at the cell membrane through interaction with FCER1G.

UniProt

FGF10 Fibroblast growth factor 10 FGF10 LungCancers Secreted (Potential). UniProt, Prediction

FGF2 Heparin-binding growth factor 2

FGF2 LungCancers, BenignNodules, Symptoms

Literature

FGF7 Keratinocyte growth factor FGF7 LungCancers, BenignNodules


FGF9 Glia-activating factor FGF9 LungCancers Secreted. UniProt, Literature, Prediction

FGFR2 Fibroblast growth factor receptor 2

FGFR2 LungCancers, BenignNodules

Cell membrane; Single-pass type I membrane protein.|Isoform 14: Secreted.|Isoform 19: Secreted.


FGFR3 Fibroblast growth factor receptor 3

FGFR3 LungCancers Membrane; Single-pass type I membrane protein.


FGL2 Fibroleukin FGL2 BenignNodules, Symptoms


FHIT Bis(5'-adenosyl)-triphosphatase FHIT LungCancers, BenignNodules, Symptoms

Cytoplasm. Literature


39





FIBA Fibrinogen alpha chain FGA LungCancers, BenignNodules, Symptoms


FINC Fibronectin FN1 Secreted, EPI, ENDO


Secreted, extracellular space, extracellular matrix.


FKB11 Peptidyl-prolyl cis-trans isomerase FKBP11

FKBP11 EPI, ENDO Membrane; Single-pass membrane protein (Potential).

UniProt, Prediction

FOLH1 Glutamate carboxypeptidase 2 FOLH1 ENDO LungCancers, Symptoms

Cell membrane; Single-pass type II membrane protein.|Isoform PSMA': Cytoplasm.

UniProt, Literature

FOLR1 Folate receptor alpha FOLR1 LungCancers Cell membrane; Lipid-anchor, GPI-anchor. Secreted (Probable).

UniProt

FOXA2 Hepatocyte nuclear factor 3-beta

FOXA2 LungCancers Nucleus. Detection, Prediction

FP100 Fanconi anemia-associated protein of 100 kDa

C17orf70 ENDO Symptoms Nucleus. Prediction

FRIH Ferritin heavy chain FTH1 EPI LungCancers, BenignNodules


FRIL Ferritin light chain FTL Secreted, EPI, ENDO

BenignNodules, Symptoms


G3P Glyceraldehyde-3-phosphate dehydrogenase

GAPDH Secreted, EPI, ENDO


Cytoplasm. Cytoplasm, perinuclear region. Membrane. Note=Postnuclear and Perinuclear regions.

Detection

G6PD Glucose-6-phosphate 1-dehydrogenase

G6PD Secreted, EPI LungCancers, Symptoms


G6PI Glucose-6-phosphate isomerase GPI Secreted, EPI Symptoms Cytoplasm. Secreted. UniProt, Literature, Detection


40





GA2L1 GAS2-like protein 1 GAS2L1 ENDO Cytoplasm, cytoskeleton (Probable). Prediction GALT2 Polypeptide N-

acetylgalactosaminyltransferase 2

GALNT2 EPI, ENDO Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein. Secreted. Note=Resides preferentially in the trans and medial parts of the Golgi stack. A secreted form also exists.

UniProt, Detection

GAS6 Growth arrest-specific protein 6 GAS6 LungCancers Secreted. UniProt, Detection, Prediction

GDIR2 Rho GDP-dissociation inhibitor 2

ARHGDIB EPI Cytoplasm. Detection

GELS Gelsolin GSN LungCancers, BenignNodules

Isoform 2: Cytoplasm, cytoskeleton.|Isoform 1: Secreted.


GGH Gamma-glutamyl hydrolase GGH LungCancers Secreted, extracellular space. Lysosome. Melanosome. Note=While its intracellular location is primarily the lysosome, most of the enzyme activity is secreted. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


GPC3 Glypican-3 GPC3 LungCancers, Symptoms

Cell membrane; Lipid-anchor, GPI-anchor; Extracellular side (By similarity).|Secreted glypican-3: Secreted, extracellular space (By similarity).


GRAN Grancalcin GCA EPI Cytoplasm. Cytoplasmic granule membrane; Peripheral membrane protein; Cytoplasmic side. Note=Primarily cytosolic in the absence of calcium or magnesium ions. Relocates to granules and other membranes in response to elevated calcium and magnesium levels.

Prediction


41





GREB1 Protein GREB1 GREB1 ENDO Membrane; Single-pass membrane protein (Potential).

UniProt, Prediction

GREM1 Gremlin-1 GREM1 LungCancers, BenignNodules

Secreted (Probable). UniProt, Prediction

GRP Gastrin-releasing peptide GRP LungCancers, Symptoms


GRP78 78 kDa glucose-regulated protein

HSPA5 Secreted, EPI, ENDO

LungCancers, BenignNodules



GSLG1 Golgi apparatus protein 1 GLG1 EPI, ENDO BenignNodules Golgi apparatus membrane; Single-pass type I membrane protein.

UniProt

GSTP1 Glutathione S-transferase P GSTP1 Secreted LungCancers, BenignNodules, Symptoms


GTR1 Solute carrier family 2, facilitated glucose transporter member 1

SLC2A1 EPI, ENDO LungCancers, BenignNodules, Symptoms

Cell membrane; Multi-pass membrane protein (By similarity). Melanosome. Note=Localizes primarily at the cell surface (By similarity). Identified by mass spectrometry in melanosome fractions from stage I to stage IV.

Literature

GTR3 Solute carrier family 2, facilitated glucose transporter member 3

SLC2A3 EPI Membrane; Multi-pass membrane protein. Detection

H2A1 Histone H2A type 1 HIST1H2AG Secreted Nucleus. Detection, Prediction

H2A1B Histone H2A type 1-B/E HIST1H2AB Secreted Nucleus. Detection, Prediction

H2A1C Histone H2A type 1-C HIST1H2AC Secreted Nucleus. Literature, Detection, Prediction


42





H2A1D Histone H2A type 1-D HIST1H2AD Secreted Nucleus. Detection, Prediction

HG2A HLA class II histocompatibility antigen gamma chain

CD74 LungCancers, BenignNodules, Symptoms

Membrane; Single-pass type II membrane protein (Potential).

UniProt, Literature

HGF Hepatocyte growth factor HGF LungCancers, BenignNodules, Symptoms


HMGA1 High mobility group protein HMG-I/HMG-Y

HMGA1 LungCancers, BenignNodules, Symptoms

Nucleus. Literature

HPRT Hypoxanthine-guanine phosphoribosyltransferase

HPRT1 EPI Cytoplasm. Detection, Prediction

HPSE Heparanase HPSE LungCancers, BenignNodules, Symptoms

Lysosome membrane; Peripheral membrane protein. Secreted. Note=Secreted, internalised and transferred to late endosomes/lysosomes as a proheparanase. In lysosomes, it is processed into the active form, the heparanase. The uptake or internalisation of proheparanase is mediated by HSPGs. Heparin appears to be a competitor and retain proheparanase in the extracellular medium.

UniProt, Prediction

HPT Haptoglobin HP LungCancers, BenignNodules, Symptoms


HS90A Heat shock protein HSP 90-alpha

HSP90AA1 Secreted, EPI LungCancers, Symptoms

Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


HS90B Heat shock protein HSP 90-beta HSP90AB1 Secreted, EPI LungCancers Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.



43





HSPB1 Heat shock protein beta-1 HSPB1 Secreted, EPI LungCancers, BenignNodules

Cytoplasm. Nucleus. Cytoplasm, cytoskeleton, spindle. Note=Cytoplasmic in interphase cells. Colocalizes with mitotic spindles in mitotic cells. Translocates to the nucleus during heat shock.


HTRA1 Serine protease HTRA1 HTRA1 LungCancers Secreted. UniProt, Prediction

HXK1 Hexokinase-1 HK1 ENDO Symptoms Mitochondrion outer membrane. Note=Its hydrophobic N-terminal sequence may be involved in membrane binding.


HYAL2 Hyaluronidase-2 HYAL2 LungCancers Cell membrane; Lipid-anchor, GPI-anchor. Prediction HYOU1 Hypoxia up-regulated protein 1 HYOU1 EPI, ENDO Symptoms Endoplasmic reticulum lumen. Detection IBP2 Insulin-like growth factor-

binding protein 2 IGFBP2 LungCancers Secreted. UniProt,


IBP3 Insulin-like growth factor-binding protein 3

IGFBP3 LungCancers, BenignNodules, Symptoms


ICAM1 Intercellular adhesion molecule 1

ICAM1 LungCancers, BenignNodules, Symptoms



ICAM3 Intercellular adhesion molecule 3

ICAM3 EPI, ENDO LungCancers, BenignNodules, Symptoms


UniProt, Detection

IDHP Isocitrate dehydrogenase [NADP], mitochondrial

IDH2 Secreted, ENDO

Mitochondrion. Prediction

IF4A1 Eukaryotic initiation factor 4A-I

EIF4A1 Secreted, EPI, ENDO



44





IGF1 Insulin-like growth factor I IGF1 LungCancers, BenignNodules, Symptoms

Secreted.|Secreted. UniProt, Literature, Detection, Prediction

IKIP Inhibitor of nuclear factor kappa-B kinase-interacting protein

IKIP ENDO Symptoms Endoplasmic reticulum membrane; Single-pass membrane protein. Note=Isoform 4 deletion of the hydrophobic, or transmembrane region between AA 45-63 results in uniform distribution troughout the cell, suggesting that this region is responsible for endoplasmic reticulum localization.

UniProt, Prediction

IL18 Interleukin-18 IL18 LungCancers, BenignNodules, Symptoms


IL19 Interleukin-19 IL19 LungCancers Secreted. UniProt, Detection, Prediction

IL22 Interleukin-22 IL22 LungCancers, BenignNodules






IL8 Interleukin-8 IL8 LungCancers, BenignNodules, Symptoms

Secreted. UniProt, Literature

ILEU Leukocyte elastase inhibitor SERPINB1 Secreted, EPI Cytoplasm (By similarity). Detection, Prediction

ILK Integrin-linked protein kinase ILK Secreted LungCancers, BenignNodules, Symptoms

Cell junction, focal adhesion. Cell membrane; Peripheral membrane protein; Cytoplasmic side.



45





INHBA Inhibin beta A chain INHBA LungCancers, BenignNodules


ISLR Immunoglobulin superfamily containing leucine-rich repeat protein

ISLR LungCancers Secreted (Potential). UniProt, Detection, Prediction

ITA5 Integrin alpha-5 ITGA5 EPI LungCancers, BenignNodules, Symptoms



ITAM Integrin alpha-M ITGAM EPI, ENDO LungCancers, BenignNodules, Symptoms


UniProt, Literature

K0090 Uncharacterized protein KIAA0090

KIAA0090 EPI Symptoms Membrane; Single-pass type I membrane protein (Potential).

UniProt, Prediction

K1C18 Keratin, type I cytoskeletal 18 KRT18 Secreted LungCancers, BenignNodules

Cytoplasm, perinuclear region. Literature, Detection, Prediction

K1C19 Keratin, type I cytoskeletal 19 KRT19 LungCancers, BenignNodules


K2C8 Keratin, type II cytoskeletal 8 KRT8 EPI LungCancers Cytoplasm. Literature, Detection

KIT Mast/stem cell growth factor receptor

KIT LungCancers Membrane; Single-pass type I membrane protein.


KITH Thymidine kinase, cytosolic TK1 LungCancers Cytoplasm. Literature, Prediction

KLK11 Kallikrein-11 KLK11 LungCancers Secreted. UniProt, Literature, Prediction


46





KLK13 Kallikrein-13 KLK13 LungCancers Secreted (Probable). UniProt, Literature, Detection, Prediction

KLK14 Kallikrein-14 KLK14 LungCancers, Symptoms

Secreted, extracellular space. UniProt, Literature, Prediction

KLK6 Kallikrein-6 KLK6 LungCancers, BenignNodules, Symptoms

Secreted. Nucleus, nucleolus. Cytoplasm. Mitochondrion. Microsome. Note=In brain, detected in the nucleus of glial cells and in the nucleus and cytoplasm of neurons. Detected in the mitochondrial and microsomal fractions of HEK-293 cells and released into the cytoplasm following cell stress.


KNG1 Kininogen-1 KNG1 LungCancers, BenignNodules, Symptoms

Secreted, extracellular space. UniProt, Detection, Prediction

KPYM Pyruvate kinase isozymes M1/M2

PKM2 Secreted, EPI LungCancers, Symptoms

Cytoplasm. Nucleus. Note=Translocates to the nucleus in response to different apoptotic stimuli. Nuclear translocation is sufficient to induce cell death that is caspase independent, isoform-specific and independent of its enzymatic actvity.


KRT35 Keratin, type I cuticular Ha5 KRT35 ENDO Detection, Prediction

LAMB2 Laminin subunit beta-2 LAMB2 ENDO LungCancers, Symptoms

Secreted, extracellular space, extracellular matrix, basement membrane. Note=S-laminin is concentrated in the synaptic cleft of the neuromuscular junction.


LDHA L-lactate dehydrogenase A chain

LDHA Secreted, EPI, ENDO

LungCancers Cytoplasm. Literature, Detection, Prediction


47





LDHB L-lactate dehydrogenase B chain

LDHB EPI LungCancers Cytoplasm. Detection, Prediction

LEG1 Galectin-1 LGALS1 Secreted LungCancers Secreted, extracellular space, extracellular matrix.

UniProt, Detection

LEG3 Galectin-3 LGALS3 LungCancers, BenignNodules

Nucleus. Note=Cytoplasmic in adenomas and carcinomas. May be secreted by a non-classical secretory pathway and associate with the cell surface.


LEG9 Galectin-9 LGALS9 ENDO Symptoms Cytoplasm (By similarity). Secreted (By similarity). Note=May also be secreted by a non-classical secretory pathway (By similarity).

UniProt

LG3BP Galectin-3-binding protein LGALS3BP Secreted LungCancers, BenignNodules, Symptoms

Secreted. Secreted, extracellular space, extracellular matrix.


LPLC3 Long palate, lung and nasal epithelium carcinoma-associated protein 3

C20orf185 LungCancers Secreted (By similarity). Cytoplasm. Note=According to PubMed:12837268 it is cytoplasmic.

UniProt, Prediction

LPLC4 Long palate, lung and nasal epithelium carcinoma-associated protein 4

C20orf186 LungCancers Secreted (By similarity). Cytoplasm. UniProt, Prediction

LPPRC Leucine-rich PPR motif-containing protein, mitochondrial

LRPPRC Secreted, ENDO

LungCancers, Symptoms

Mitochondrion. Nucleus, nucleoplasm. Nucleus inner membrane. Nucleus outer membrane. Note=Seems to be predominantly mitochondrial.

Prediction


48





LRP1 Prolow-density lipoprotein receptor-related protein 1

LRP1 EPI LungCancers, Symptoms

Low-density lipoprotein receptor-related protein 1 85 kDa subunit: Cell membrane; Single-pass type I membrane protein. Membrane, coated pit.|Low-density lipoprotein receptor-related protein 1 515 kDa subunit: Cell membrane; Peripheral membrane protein; Extracellular side. Membrane, coated pit.|Low-density lipoprotein receptor-related protein 1 intracellular domain: Cytoplasm. Nucleus. Note=After cleavage, the intracellular domain (LRPICD) is detected both in the cytoplasm and in the nucleus.

UniProt, Detection

LUM Lumican LUM Secreted, EPI LungCancers, BenignNodules, Symptoms



LY6K Lymphocyte antigen 6K LY6K LungCancers, Symptoms

Secreted. Cytoplasm. Cell membrane; Lipid- anchor, GPI-anchor (Potential).

UniProt, Prediction

LYAM2 E-selectin SELE LungCancers, BenignNodules, Symptoms



LYAM3 P-selectin SELP LungCancers, BenignNodules, Symptoms



LYOX Protein-lysine 6-oxidase LOX LungCancers, BenignNodules

Secreted, extracellular space. UniProt, Detection, Prediction

LYPD3 Ly6/PLAUR domain-containing protein 3

LYPD3 LungCancers Cell membrane; Lipid-anchor, GPI-anchor. Detection, Prediction

MAGA4 Melanoma-associated antigen 4 MAGEA4 LungCancers Literature, Prediction


49





MASP1 Mannan-binding lectin serine protease 1

MASP1 LungCancers, Symptoms


MDHC Malate dehydrogenase, cytoplasmic

MDH1 Secreted Cytoplasm. Literature, Detection, Prediction

MDHM Malate dehydrogenase, mitochondrial

MDH2 ENDO LungCancers Mitochondrion matrix. Detection, Prediction

MIF Macrophage migration inhibitory factor

MIF Secreted LungCancers, BenignNodules, Symptoms

Secreted. Cytoplasm. Note=Does not have a cleavable signal sequence and is secreted via a specialized, non- classical pathway. Secreted by macrophages upon stimulation by bacterial lipopolysaccharide (LPS), or by M.tuberculosis antigens.


MLH1 DNA mismatch repair protein Mlh1

MLH1 ENDO LungCancers, BenignNodules, Symptoms

Nucleus. Literature

MMP1 Interstitial collagenase MMP1 LungCancers, BenignNodules, Symptoms

Secreted, extracellular space, extracellular matrix (Probable).


MMP11 Stromelysin-3 MMP11 LungCancers, Symptoms



MMP12 Macrophage metalloelastase MMP12 LungCancers, BenignNodules, Symptoms



MMP14 Matrix metalloproteinase-14 MMP14 ENDO LungCancers, BenignNodules, Symptoms

Membrane; Single-pass type I membrane protein (Potential). Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.



50





MMP2 72 kDa type IV collagenase MMP2 LungCancers, BenignNodules, Symptoms



MMP26 Matrix metalloproteinase-26 MMP26 LungCancers Secreted, extracellular space, extracellular matrix.

UniProt, Prediction

MMP7 Matrilysin MMP7 LungCancers, BenignNodules, Symptoms



MMP9 Matrix metalloproteinase-9 MMP9 LungCancers, BenignNodules, Symptoms



MOGS Mannosyl-oligosaccharide glucosidase

MOGS ENDO Endoplasmic reticulum membrane; Single-pass type II membrane protein.

UniProt, Prediction

MPRI Cation-independent mannose-6-phosphate receptor

IGF2R EPI, ENDO LungCancers, Symptoms

Lysosome membrane; Single-pass type I membrane protein.


MRP3 Canalicular multispecific organic anion transporter 2

ABCC3 EPI LungCancers Membrane; Multi-pass membrane protein. Literature, Detection


51





MUC1 Mucin-1 MUC1 EPI LungCancers, BenignNodules, Symptoms

Apical cell membrane; Single-pass type I membrane protein. Note=Exclusively located in the apical domain of the plasma membrane of highly polarized epithelial cells. After endocytosis, internalized and recycled to the cell membrane. Located to microvilli and to the tips of long filopodial protusions.|Isoform 5: Secreted.|Isoform 7: Secreted.|Isoform 9: Secreted.|Mucin-1 subunit beta: Cell membrane. Cytoplasm. Nucleus. Note=On EGF and PDGFRB stimulation, transported to the nucleus through interaction with CTNNB1, a process which is stimulated by phosphorylation. On HRG stimulation, colocalizes with JUP/gamma-catenin at the nucleus.


MUC16 Mucin-16 MUC16 LungCancers Cell membrane; Single-pass type I membrane protein. Secreted, extracellular space. Note=May be liberated into the extracellular space following the phosphorylation of the intracellular C-terminus which induces the proteolytic cleavage and liberation of the extracellular domain.

UniProt, Detection


52





MUC4 Mucin-4 MUC4 LungCancers, BenignNodules

Membrane; Single-pass membrane protein (Potential). Secreted. Note=Isoforms lacking the Cys-rich region, EGF-like domains and transmembrane region are secreted. Secretion occurs by splicing or proteolytic processing.|Mucin-4 beta chain: Cell membrane; Single- pass membrane protein.|Mucin-4 alpha chain: Secreted.|Isoform 3: Cell membrane; Single-pass membrane protein.|Isoform 15: Secreted.

UniProt

MUC5B Mucin-5B MUC5B LungCancers, BenignNodules


MUCL1 Mucin-like protein 1 MUCL1 LungCancers Secreted (Probable). Membrane (Probable). UniProt, Prediction

NAMPT Nicotinamide phosphoribosyltransferase

NAMPT EPI LungCancers, BenignNodules, Symptoms

Cytoplasm (By similarity). Literature, Detection

NAPSA Napsin-A NAPSA Secreted LungCancers Prediction NCF4 Neutrophil cytosol factor 4 NCF4 ENDO Cytoplasm. Prediction NDKA Nucleoside diphosphate kinase

A NME1 Secreted LungCancers,

BenignNodules, Symptoms

Cytoplasm. Nucleus. Note=Cell-cycle dependent nuclear localization which can be induced by interaction with Epstein-barr viral proteins or by degradation of the SET complex by GzmA.


NDKB Nucleoside diphosphate kinase B

NME2 Secreted, EPI BenignNodules Cytoplasm. Nucleus. Note=Isoform 2 is mainly cytoplasmic and isoform 1 and isoform 2 are excluded from the nucleolus.


NDUS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial

NDUFS1 Secreted, ENDO

Symptoms Mitochondrion inner membrane. Prediction

NEBL Nebulette NEBL ENDO Prediction


53





NEK4 Serine/threonine-protein kinase Nek4

NEK4 ENDO LungCancers Nucleus (Probable). Prediction

NET1 Netrin-1 NTN1 LungCancers, BenignNodules



NEU2 Vasopressin-neurophysin 2-copeptin

AVP LungCancers, Symptoms


NGAL Neutrophil gelatinase-associated lipocalin

LCN2 EPI LungCancers, BenignNodules, Symptoms


NGLY1 Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase

NGLY1 ENDO Cytoplasm. Detection, Prediction

NHRF1 Na(+)/H(+) exchange regulatory cofactor NHE-RF1

SLC9A3R1 EPI BenignNodules Endomembrane system; Peripheral membrane protein. Cell projection, filopodium. Cell projection, ruffle. Cell projection, microvillus. Note=Colocalizes with actin in microvilli-rich apical regions of the syncytiotrophoblast. Found in microvilli, ruffling membrane and filopodia of HeLa cells. Present in lipid rafts of T-cells.

Detection

NIBAN Protein Niban FAM129A EPI Cytoplasm. Literature, Detection

NMU Neuromedin-U NMU LungCancers Secreted. UniProt, Prediction

NRP1 Neuropilin-1 NRP1 LungCancers, BenignNodules, Symptoms

Cell membrane; Single-pass type I membrane protein.|Isoform 2: Secreted.


ODAM Odontogenic ameloblast-associated protein

ODAM LungCancers Secreted (By similarity). UniProt, Prediction


54





OSTP Osteopontin SPP1 LungCancers, BenignNodules, Symptoms


OVOS2 Ovostatin homolog 2 OVOS2 ENDO Secreted (By similarity). UniProt, Prediction

P5CS Delta-1-pyrroline-5-carboxylate synthase

ALDH18A1 ENDO Mitochondrion inner membrane. Prediction

PA2GX Group 10 secretory phospholipase A2

PLA2G10 Symptoms Secreted. UniProt

PAPP1 Pappalysin-1 PAPPA LungCancers, BenignNodules, Symptoms


PBIP1 Pre-B-cell leukemia transcription factor-interacting protein 1

PBXIP1 EPI Cytoplasm, cytoskeleton. Nucleus. Note=Shuttles between the nucleus and the cytosol. Mainly localized in the cytoplasm, associated with microtubules. Detected in small amounts in the nucleus.

Prediction

PCBP1 Poly(rC)-binding protein 1 PCBP1 EPI, ENDO Nucleus. Cytoplasm. Note=Loosely bound in the nucleus. May shuttle between the nucleus and the cytoplasm.


PCBP2 Poly(rC)-binding protein 2 PCBP2 EPI Nucleus. Cytoplasm. Note=Loosely bound in the nucleus. May shuttle between the nucleus and the cytoplasm.


PCD15 Protocadherin-15 PCDH15 ENDO Cell membrane; Single-pass type I membrane protein (By similarity).|Isoform 3: Secreted.

UniProt, Detection

PCNA Proliferating cell nuclear antigen

PCNA EPI LungCancers, BenignNodules, Symptoms

Nucleus. Literature, Prediction

PCYOX Prenylcysteine oxidase 1 PCYOX1 Secreted LungCancers, Symptoms

Lysosome. Detection, Prediction


55





PDGFA Platelet-derived growth factor subunit A

PDGFA LungCancers Secreted. UniProt, Literature, Prediction

PDGFB Platelet-derived growth factor subunit B

PDGFB LungCancers, BenignNodules, Symptoms


PDGFD Platelet-derived growth factor D

PDGFD LungCancers Secreted. UniProt, Prediction

PDIA3 Protein disulfide-isomerase A3 PDIA3 ENDO LungCancers Endoplasmic reticulum lumen (By similarity). Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


PDIA4 Protein disulfide-isomerase A4 PDIA4 Secreted, EPI, ENDO



PDIA6 Protein disulfide-isomerase A6 PDIA6 Secreted, EPI, ENDO

Endoplasmic reticulum lumen (By similarity). Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


PECA1 Platelet endothelial cell adhesion molecule

PECAM1 LungCancers, BenignNodules, Symptoms



PEDF Pigment epithelium-derived factor

SERPINF1 LungCancers, Symptoms

Secreted. Melanosome. Note=Enriched in stage I melanosomes.


PERM Myeloperoxidase MPO Secreted, EPI, ENDO


Lysosome. Literature, Detection, Prediction


56





PERP1 Plasma cell-induced resident endoplasmic reticulum protein

PACAP EPI, ENDO Secreted (Potential). Cytoplasm. Note=In (PubMed:11350957) diffuse granular localization in the cytoplasm surrounding the nucleus.


PGAM1 Phosphoglycerate mutase 1 PGAM1 Secreted, EPI LungCancers, Symptoms

Detection

PLAC1 Placenta-specific protein 1 PLAC1 LungCancers Secreted (Probable). UniProt, Prediction

PLACL Placenta-specific 1-like protein PLAC1L LungCancers Secreted (Potential). UniProt, Prediction

PLIN2 Perilipin-2 ADFP ENDO LungCancers Membrane; Peripheral membrane protein. Prediction PLIN3 Perilipin-3 M6PRBP1 EPI Cytoplasm. Endosome membrane;

Peripheral membrane protein; Cytoplasmic side (Potential). Lipid droplet (Potential). Note=Membrane associated on endosomes. Detected in the envelope and the core of lipid bodies and in lipid sails.


PLOD1 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1

PLOD1 EPI, ENDO Rough endoplasmic reticulum membrane; Peripheral membrane protein; Lumenal side.

Prediction

PLOD2 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2

PLOD2 ENDO BenignNodules, Symptoms

Rough endoplasmic reticulum membrane; Peripheral membrane protein; Lumenal side.

Prediction


57





PLSL Plastin-2 LCP1 Secreted, EPI LungCancers Cytoplasm, cytoskeleton. Cell junction. Cell projection. Cell projection, ruffle membrane; Peripheral membrane protein; Cytoplasmic side (By similarity). Note=Relocalizes to the immunological synapse between peripheral blood T lymphocytes and antibody-presenting cells in response to costimulation through TCR/CD3 and CD2 or CD28. Associated with the actin cytoskeleton at membrane ruffles (By similarity). Relocalizes to actin-rich cell projections upon serine phosphorylation.


PLUNC Protein Plunc PLUNC LungCancers, BenignNodules

Secreted (By similarity). Note=Found in the nasal mucus (By similarity). Apical side of airway epithelial cells. Detected in nasal mucus (By similarity).

UniProt, Prediction

PLXB3 Plexin-B3 PLXNB3 ENDO Membrane; Single-pass type I membrane protein.


PLXC1 Plexin-C1 PLXNC1 EPI Membrane; Single-pass type I membrane protein (Potential).

UniProt, Detection

POSTN Periostin POSTN Secreted, ENDO




PPAL Lysosomal acid phosphatase ACP2 EPI Symptoms Lysosome membrane; Single-pass membrane protein; Lumenal side. Lysosome lumen. Note=The soluble form arises by proteolytic processing of the membrane-bound form.

UniProt, Prediction


58





PPBT Alkaline phosphatase, tissue-nonspecific isozyme

ALPL EPI LungCancers, BenignNodules, Symptoms

Cell membrane; Lipid-anchor, GPI-anchor. Literature, Detection, Prediction

PPIB Peptidyl-prolyl cis-trans isomerase B

PPIB Secreted, EPI, ENDO



PRDX1 Peroxiredoxin-1 PRDX1 EPI LungCancers Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


PRDX4 Peroxiredoxin-4 PRDX4 Secreted, EPI, ENDO

Cytoplasm. Literature, Detection, Prediction

PROF1 Profilin-1 PFN1 Secreted, EPI LungCancers Cytoplasm, cytoskeleton. Detection PRP31 U4/U6 small nuclear

ribonucleoprotein Prp31 PRPF31 ENDO Nucleus speckle. Nucleus, Cajal body.

Note=Predominantly found in speckles and in Cajal bodies.

Prediction

PRS6A 26S protease regulatory subunit 6A

PSMC3 EPI BenignNodules Cytoplasm (Potential). Nucleus (Potential). Detection

PSCA Prostate stem cell antigen PSCA LungCancers Cell membrane; Lipid-anchor, GPI-anchor. Literature, Prediction

PTGIS Prostacyclin synthase PTGIS EPI LungCancers, BenignNodules

Endoplasmic reticulum membrane; Single-pass membrane protein.


PTPA Serine/threonine-protein phosphatase 2A activator

PPP2R4 ENDO Symptoms Detection, Prediction

PTPRC Receptor-type tyrosine-protein phosphatase C

PTPRC Secreted, EPI, ENDO

LungCancers Membrane; Single-pass type I membrane protein.


PTPRJ Receptor-type tyrosine-protein phosphatase eta

PTPRJ EPI LungCancers, Symptoms




59





PVR Poliovirus receptor PVR Symptoms Isoform Alpha: Cell membrane; Single-pass type I membrane protein.|Isoform Delta: Cell membrane; Single-pass type I membrane protein.|Isoform Beta: Secreted.|Isoform Gamma: Secreted.


RAB32 Ras-related protein Rab-32 RAB32 EPI Mitochondrion. Prediction RAGE Advanced glycosylation end

product-specific receptor AGER Secreted LungCancers,

BenignNodules Isoform 1: Cell membrane; Single-pass type I membrane protein.|Isoform 2: Secreted.

UniProt, Literature

RAN GTP-binding nuclear protein Ran

RAN Secreted, EPI LungCancers, BenignNodules

Nucleus. Cytoplasm. Melanosome. Note=Becomes dispersed throughout the cytoplasm during mitosis. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


RAP2B Ras-related protein Rap-2b RAP2B EPI Cell membrane; Lipid-anchor; Cytoplasmic side (Potential).

Prediction

RAP2C Ras-related protein Rap-2c RAP2C EPI Cell membrane; Lipid-anchor; Cytoplasmic side (Potential).

Prediction

RCN3 Reticulocalbin-3 RCN3 EPI Symptoms Endoplasmic reticulum lumen (Potential). Prediction RL24 60S ribosomal protein L24 RPL24 EPI Prediction S10A1 Protein S100-A1 S100A1 Symptoms Cytoplasm. Literature,

Prediction S10A6 Protein S100-A6 S100A6 Secreted LungCancers Nucleus envelope. Cytoplasm. Literature,


S10A7 Protein S100-A7 S100A7 LungCancers Cytoplasm. Secreted. Note=Secreted by a non- classical secretory pathway.


SAA Serum amyloid A protein SAA1 Symptoms Secreted. UniProt, Literature, Detection, Prediction


60





SCF Kit ligand KITLG LungCancers, Symptoms

Isoform 1: Cell membrane; Single-pass type I membrane protein (By similarity). Secreted (By similarity). Note=Also exists as a secreted soluble form (isoform 1 only) (By similarity).|Isoform 2: Cell membrane; Single-pass type I membrane protein (By similarity). Cytoplasm, cytoskeleton (By similarity).

UniProt, Literature

SDC1 Syndecan-1 SDC1 LungCancers, BenignNodules, Symptoms



SEM3G Semaphorin-3G SEMA3G LungCancers Secreted (By similarity). UniProt, Prediction

SEPR Seprase FAP ENDO Symptoms Cell membrane; Single-pass type II membrane protein. Cell projection, lamellipodium membrane; Single-pass type II membrane protein. Cell projection, invadopodium membrane; Single-pass type II membrane protein. Note=Found in cell surface lamellipodia, invadopodia and on shed vesicles.


SERPH Serpin H1 SERPINH1 Secreted, EPI, ENDO

LungCancers, BenignNodules

Endoplasmic reticulum lumen. Detection, Prediction

SFPA2 Pulmonary surfactant-associated protein A2

SFTPA2 Secreted LungCancers, BenignNodules

Secreted, extracellular space, extracellular matrix. Secreted, extracellular space, surface film.

UniProt, Prediction

SFTA1 Pulmonary surfactant-associated protein A1

SFTPA1 Secreted LungCancers, BenignNodules, Symptoms

Secreted, extracellular space, extracellular matrix. Secreted, extracellular space, surface film.

UniProt, Prediction

SG3A2 Secretoglobin family 3A member 2

SCGB3A2 LungCancers, BenignNodules


SGPL1 Sphingosine-1-phosphate lyase 1

SGPL1 ENDO Endoplasmic reticulum membrane; Single-pass type III membrane protein.

UniProt, Prediction


61





SIAL Bone sialoprotein 2 IBSP LungCancers Secreted. UniProt, Literature, Prediction

SLPI Antileukoproteinase SLPI LungCancers, BenignNodules


SMD3 Small nuclear ribonucleoprotein Sm D3

SNRPD3 Secreted BenignNodules Nucleus. Prediction

SMS Somatostatin SST LungCancers Secreted. UniProt, Literature, Prediction

SODM Superoxide dismutase [Mn], mitochondrial

SOD2 Secreted LungCancers, BenignNodules, Symptoms

Mitochondrion matrix. Literature, Detection, Prediction

SORL Sortilin-related receptor SORL1 EPI LungCancers, Symptoms

Membrane; Single-pass type I membrane protein (Potential).

UniProt, Detection

SPB3 Serpin B3 SERPINB3 LungCancers, BenignNodules

Cytoplasm. Note=Seems to also be secreted in plasma by cancerous cells but at a low level.


SPB5 Serpin B5 SERPINB5 LungCancers Secreted, extracellular space. UniProt, Detection

SPON2 Spondin-2 SPON2 LungCancers, BenignNodules


UniProt, Prediction

SPRC SPARC SPARC LungCancers, BenignNodules, Symptoms

Secreted, extracellular space, extracellular matrix, basement membrane. Note=In or around the basement membrane.


SRC Proto-oncogene tyrosine-protein kinase Src

SRC ENDO LungCancers, BenignNodules, Symptoms

Literature


62





SSRD Translocon-associated protein subunit delta

SSR4 Secreted, ENDO

Endoplasmic reticulum membrane; Single-pass type I membrane protein.

UniProt, Prediction

STAT1 Signal transducer and activator of transcription 1-alpha/beta

STAT1 EPI LungCancers, BenignNodules

Cytoplasm. Nucleus. Note=Translocated into the nucleus in response to IFN-gamma-induced tyrosine phosphorylation and dimerization.

Detection

STAT3 Signal transducer and activator of transcription 3

STAT3 ENDO LungCancers, BenignNodules, Symptoms

Cytoplasm. Nucleus. Note=Shuttles between the nucleus and the cytoplasm. Constitutive nuclear presence is independent of tyrosine phosphorylation.

Prediction

STC1 Stanniocalcin-1 STC1 LungCancers, Symptoms


STT3A Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A

STT3A EPI Symptoms Endoplasmic reticulum membrane; Multi-pass membrane protein.

Literature

TAGL Transgelin TAGLN EPI LungCancers Cytoplasm (Probable). Literature, Prediction

TARA TRIO and F-actin-binding protein

TRIOBP ENDO Nucleus. Cytoplasm, cytoskeleton. Note=Localized to F-actin in a periodic pattern.


TBA1B Tubulin alpha-1B chain TUBA1B EPI LungCancers Detection TBB2A Tubulin beta-2A chain TUBB2A EPI LungCancers,

BenignNodules Detection,

Prediction TBB3 Tubulin beta-3 chain TUBB3 EPI LungCancers,

BenignNodules Detection

TBB5 Tubulin beta chain TUBB EPI LungCancers, BenignNodules

Detection

TCPA T-complex protein 1 subunit alpha

TCP1 EPI Cytoplasm. Prediction


63





TCPD T-complex protein 1 subunit delta

CCT4 EPI Cytoplasm. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.


TCPQ T-complex protein 1 subunit theta

CCT8 Secreted, EPI Cytoplasm. Prediction

TCPZ T-complex protein 1 subunit zeta

CCT6A Secreted, EPI Cytoplasm. Detection

TDRD3 Tudor domain-containing protein 3

TDRD3 ENDO Cytoplasm. Nucleus. Note=Predominantly cytoplasmic. Associated with actively translating polyribosomes and with mRNA stress granules.

Prediction

TENA Tenascin TNC ENDO LungCancers, BenignNodules, Symptoms



TENX Tenascin-X TNXB ENDO LungCancers, Symptoms



TERA Transitional endoplasmic reticulum ATPase

VCP EPI LungCancers, BenignNodules

Cytoplasm, cytosol. Nucleus. Note=Present in the neuronal hyaline inclusion bodies specifically found in motor neurons from amyotrophic lateral sclerosis patients. Present in the Lewy bodies specifically found in neurons from Parkinson disease patients.

Detection

TETN Tetranectin CLEC3B LungCancers Secreted. UniProt, Literature, Detection, Prediction

TF Tissue factor F3 LungCancers, BenignNodules, Symptoms


UniProt, Literature


64





TFR1 Transferrin receptor protein 1 TFRC Secreted, EPI, ENDO


Cell membrane; Single-pass type II membrane protein. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.|Transferrin receptor protein 1, serum form: Secreted.


TGFA Protransforming growth factor alpha

TGFA LungCancers, BenignNodules

Transforming growth factor alpha: Secreted, extracellular space.|Protransforming growth factor alpha: Cell membrane; Single-pass type I membrane protein.

UniProt, Literature

THAS Thromboxane-A synthase TBXAS1 EPI, ENDO LungCancers, BenignNodules, Symptoms

Membrane; Multi-pass membrane protein. Prediction

THY1 Thy-1 membrane glycoprotein THY1 EPI Symptoms Cell membrane; Lipid-anchor, GPI-anchor (By similarity).


TIMP1 Metalloproteinase inhibitor 1 TIMP1 LungCancers, BenignNodules, Symptoms


TIMP3 Metalloproteinase inhibitor 3 TIMP3 LungCancers, BenignNodules



TLL1 Tolloid-like protein 1 TLL1 ENDO Secreted (Probable). UniProt, Prediction

TNF12 Tumor necrosis factor ligand superfamily member 12

TNFSF12 LungCancers, BenignNodules

Cell membrane; Single-pass type II membrane protein.|Tumor necrosis factor ligand superfamily member 12, secreted form: Secreted.

UniProt


65





TNR6 Tumor necrosis factor receptor superfamily member 6

FAS LungCancers, BenignNodules, Symptoms

Isoform 1: Cell membrane; Single-pass type I membrane protein.|Isoform 2: Secreted.|Isoform 3: Secreted.|Isoform 4: Secreted.|Isoform 5: Secreted.|Isoform 6: Secreted.


TPIS Triosephosphate isomerase TPI1 Secreted, EPI Symptoms Literature, Detection, Prediction

TRFL Lactotransferrin LTF Secreted, EPI, ENDO



TSP1 Thrombospondin-1 THBS1 LungCancers, BenignNodules, Symptoms


TTHY Transthyretin TTR LungCancers, BenignNodules

Secreted. Cytoplasm. UniProt, Literature, Detection, Prediction

TYPH Thymidine phosphorylase TYMP EPI LungCancers, BenignNodules, Symptoms


UGGG1 UDP-glucose:glycoprotein glucosyltransferase 1

UGGT1 Secreted, ENDO

Endoplasmic reticulum lumen. Endoplasmic reticulum-Golgi intermediate compartment.


UGGG2 UDP-glucose:glycoprotein glucosyltransferase 2

UGGT2 ENDO Endoplasmic reticulum lumen. Endoplasmic reticulum-Golgi intermediate compartment.

Prediction

UGPA UTP--glucose-1-phosphate uridylyltransferase

UGP2 EPI Symptoms Cytoplasm. Detection

UPAR Urokinase plasminogen activator surface receptor

PLAUR LungCancers, BenignNodules, Symptoms

Isoform 1: Cell membrane; Lipid-anchor, GPI- anchor.|Isoform 2: Secreted (Probable).



66





UTER Uteroglobin SCGB1A1 LungCancers, BenignNodules, Symptoms


VA0D1 V-type proton ATPase subunit d 1

ATP6V0D1 EPI Prediction

VAV3 Guanine nucleotide exchange factor VAV3

VAV3 ENDO Prediction

VEGFA Vascular endothelial growth factor A

VEGFA LungCancers, BenignNodules, Symptoms

Secreted. Note=VEGF121 is acidic and freely secreted. VEGF165 is more basic, has heparin-binding properties and, although a signicant proportion remains cell-associated, most is freely secreted. VEGF189 is very basic, it is cell-associated after secretion and is bound avidly by heparin and the extracellular matrix, although it may be released as a soluble form by heparin, heparinase or plasmin.


VEGFC Vascular endothelial growth factor C

VEGFC LungCancers, BenignNodules


VEGFD Vascular endothelial growth factor D

FIGF LungCancers Secreted. UniProt, Literature, Prediction

VGFR1 Vascular endothelial growth factor receptor 1

FLT1 LungCancers, BenignNodules, Symptoms

Isoform Flt1: Cell membrane; Single-pass type I membrane protein.|Isoform sFlt1: Secreted.


VTNC Vitronectin VTN ENDO Symptoms Secreted, extracellular space. UniProt, Literature, Detection, Prediction


67





VWC2 Brorin VWC2 LungCancers Secreted, extracellular space, extracellular matrix, basement membrane (By similarity).

UniProt, Prediction

WNT3A Protein Wnt-3a WNT3A LungCancers, Symptoms


UniProt, Prediction

WT1 Wilms tumor protein WT1 LungCancers, BenignNodules, Symptoms

Nucleus. Cytoplasm (By similarity). Note=Shuttles between nucleus and cytoplasm (By similarity).|Isoform 1: Nucleus speckle.|Isoform 4: Nucleus, nucleoplasm.


ZA2G Zinc-alpha-2-glycoprotein AZGP1 LungCancers, Symptoms


ZG16B Zymogen granule protein 16 homolog B

ZG16B LungCancers Secreted (Potential). UniProt, Prediction

Note: • Source of tissue biomarkers

Datasets from which the protein was identified as a tissue biomarker: Secreted (secretory vesicles of 33 lung cancer patients, including 13 adenocarcinoma, 5 large cell carcinoma and 15 squamous cell carcinoma), EPI (membrane vesicles of epithelial cells of 30 lung cancer patients, including 19 adenocarcinoma, 5 large cell carcinoma, and 6 squamous cell carcinoma), and ENDO (membrane vesicles of endothelial cells of 38 lung cancer patients, including 13 adenocarcinoma, 7 large cell carcinoma, and 18 squamous cell carcinoma): See Table S1.

• Biomarkers in literature Terminologies with which the protein or the corresponding gene was associated in the literature: LungCancers (subtypes of lung cancers), BenignNodules (diseases causing benign lung nodules), and Symptoms (symptoms of benign or cancerous lung nodules): See Table S2.

• Evidence for presence in blood


68

Evidence for presence in blood based on four sources: UniProt (annotated as a secreted protein or a single-pass transmembrane protein), Literature (57), Detection (previously detected in blood samples by mass spectrometry (54-56)), or Prediction (predicted as a secreted protein by TMHMM (58), SignalP (59), and/or SecretomeP (60)).


69

Table S4. Summary of protein MRM assay development and detection in blood.

Characteristic Selected for assay development

Developed MRM assays

Success rate (%)

Detected in blood

Detection rate (%)

Source of candidates

Tissue biomarkers & biomarkers in literature

148 146 98.6 91 62.3

Tissue biomarkers only 69 69 100.0 23 33.3 Biomarkers in literature only 171 156 91.2 76 48.7 All tissue biomarkers 217 215 99.1 114 53.0 All biomarkers in literature 319 302 94.7 167 55.3 Evidence for presence in blood (see Table S3) UniProt, Literature, Detection & Prediction

42 39 92.9 33 84.6

UniProt, Literature & Detection 18 18 100.0 11 61.1 UniProt, Literature & Prediction 34 32 94.1 12 37.5 UniProt, Detection & Prediction 30 30 100.0 14 46.7 Literature, Detection & Prediction

24 24 100.0 15 62.5

UniProt & Literature 13 13 100.0 7 53.8 UniProt & Detection 16 16 100.0 11 68.8 UniProt & Prediction 55 47 85.5 15 31.9 Literature & Detection 22 22 100.0 10 45.5 Literature & Prediction 10 10 100.0 4 40.0 Detection & Prediction 40 40 100.0 23 57.5 UniProt only 12 10 83.3 5 50.0 Literature only 10 9 90.0 4 44.4 Detection only 22 22 100.0 17 77.3 Prediction only 40 39 97.5 9 23.1 All with UniProt 220 205 93.2 108 52.7 All with Literature 173 167 96.5 96 57.5 All with Detection 214 211 98.6 134 63.5 All with Prediction 275 261 94.9 125 47.9 Overall 388 371 95.6 190 51.2


70

Table S5. MRM assay of individual proteins.

(In a separate file of 277 pages: See (17) for definition of terminologies.)


71

Table S6. Experimental design of the discovery study.

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MXCC05430H11101XMR1B

1 1 HPS HPS NA NA NA NA NA NA NA NA 0.012

MZCO58030H11101XMR1

1 2 IUCPQ 2683 81 Male 10 Past 62.5 Benign Granuloma Necrotizing Granuloma 0.860

MZCO55530H11101XMR1

1 3 IUCPQ 1996 78 Male 10 Past NA Cancer Adenocarcinoma NA 0.371

MZCO59230H11101XMR1

1 4 IUCPQ 2984 77 Male 26 NA 45.6 Benign Hamartoma NA 0.505

MZCO57530H11101XMR1

1 5 IUCPQ 2585 75 Male 9 Past 91.5 Cancer Adenocarcinoma NA 0.915

MZCO55930H11101XMR1

1 6 IUCPQ 2042 56 Female 13 Past 39 Cancer Adenocarcinoma NA 0.857

MZCO58130H11101XMR1

1 7 IUCPQ 2690 61 Female 14 Never 0 Benign Granuloma Necrotizing Granuloma 0.092

MZCO58930H11101XMR1

1 8 IUCPQ 2912 62 Male 13 Past 25 Cancer Adenocarcinoma NA 0.038

MZCO58730H11101XMR1

1 9 IUCPQ 2866 63 Male 10 Current 36 Benign Granuloma Necrotizing Granuloma 0.672

MZCO55630H11101XMR1

1 10 IUCPQ 1999 42 Female 12 Never 0 Benign Hamartoma Hamartoma 0.625

MZCO59030H11101XMR1


MZCO56330H11101XMR1


MZCO59630H11101XMR1

1 13 IUCPQ 3242 48 Male 13 Current 13.2 Benign Granuloma Necrotizing Granuloma 0.793

MZCO56730H11101XMR1

1 14 IUCPQ 2331 78 Male 9 Past 19.5 Benign Other Pulmonary Infarcct 0.801

MZCO57730H11101XMR1



72


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MXCC05430H11101XMR5


MZCO57930H11101XMR1

1 17 IUCPQ 2680 62 Male 40 Past NA Benign Other Hemorraghic Infarct 0.435

MZCO59430H11101XMR1


MZCO58430H11101XMR1

1 19 IUCPQ 2796 64 Male 14 Past 42 Benign Granuloma Necrotizing Granuloma 0.329

MZCO56030H11101XMR1

1 20 IUCPQ 2071 66 Male 13 Past NA Cancer Adenocarcinoma NA 0.834

MZCO56130H11101XMR1


MZCO59930H11101XMR1

1 22 IUCPQ 3512 52 Female 10 Current 39 Benign Granuloma Necrotizing Granuloma 0.886

MZCO59530H11101XMR1

1 23 IUCPQ 3239 66 Male 12 Current NA Benign Other Fibroelastic Nodule 0.617

MZCO56830H11101XMR1


MZCO57030H11101XMR1

1 25 IUCPQ 2370 49 Female 10 Current 15 Cancer Adenocarcinoma NA 0.876

MZCO55030H11101XMR1

1 26 IUCPQ 1845 52 Female 9 Past 40 Benign Hamartoma NA 0.483

MZCO57130H11101XMR1

1 27 IUCPQ 2419 65 Male 5 Past 55 Benign Granuloma Necrotizing Granuloma 0.498

MZCO59130H11101XMR1


MZCO58530H11101XMR1

1 29 IUCPQ 2806 55 Male 17 Current 40 Cancer Adenocarcinoma NA 0.871

MZCO58330H11101XMR1

1 30 IUCPQ 2789 47 Male 12 Current 34 Benign Other Amyloidosis 0.148


73


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MXCC05430H11101XMR6


MXCC05431H11101XMR1


MZCO53631H11101XMR1

2 2 UPenn 20574 64 Female 39 Past 7.5 Cancer Adenocarcinoma NA 0.789

MZCO48531H11101XMR1

2 3 UPenn 20302 63 Female 35 Never 0 Benign Granuloma NA 0.416

MZCO51831H11101XMR1

2 4 UPenn 20490 63 Female 18 Past 45 Benign Granuloma Necrotizing Granuloma 0.698

MZCO38531H11101XMR1

2 5 UPenn 1416 56 Male 28 Past 40 Cancer Adenocarcinoma NA 0.951

MZCO43031H11101XMR1

2 6 UPenn 20038 65 Female 9 Past 20 Cancer Adenocarcinoma NA 0.433

MZCO51631H11101XMR1

2 7 UPenn 20475 65 Female 7 Never 0 Benign Granuloma Non-necrotizing Granuloma 0.400

MZCO36931H11101XMR1

2 8 UPenn 1289 52 Male 13 Past 50 Benign Granuloma Necrotizing Granuloma 0.570

MZCO54231H11101XMR1


MZCO47931H11101XMR1

2 10 UPenn 20281 68 Female 45 Never 0 Cancer Adenocarcinoma NA 0.512

MZCO49831H11101XMR1

2 11 UPenn 20392 70 Female 12 Never 0 Benign Granuloma Necrotizing Granuloma 0.317

MZCO48231H11101XMR1

2 12 UPenn 20290 69 Male 13 Past 60 Benign Other Amyloidosis 0.557

MZCO37731H11101XMR1

2 13 UPenn 1344 69 Male 20 Past 120 Cancer Other Large Cell Squamous Cell 0.548

MZCO53131H11101XMR1



74


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MZCO42231H11101XMR1

2 15 UPenn 1755 77 Female 20 Past 8.6 Benign Scar NA 0.821

MXCC05431H11101XMR2


MZCO45131H11101XMR1

2 17 UPenn 20150 44 Female 11.6 Past 6 Benign Hamartoma Hamartoma 0.059

MZCO38831H11101XMR1


MZCO38431H11101XMR1

2 19 UPenn 1403 68 Male 14 Past 4.5 Cancer Mixed NA 0.909

MZCO49931H11101XMR1

2 20 UPenn 20403 72 Male 1.5 Past 15 Benign Granuloma Necrotizing Granuloma 0.510

MZCO46031H11101XMR1


MZCO37131H11101XMR1

2 22 UPenn 1305 57 Female 10 Past 20 Benign Granuloma NA 0.018

MZCO47431H11101XMR1

2 23 UPenn 20250 44 Male 15 Past 45 Benign Hamartoma NA 0.289

MZCO39231H11101XMR1


MZCO44031H11101XMR1


MZCO37231H11101XMR1

2 26 UPenn 1309 57 Female 30 NA 60 Benign Other Subpleural Fibrosis Benign Lymphoid Hyperplasia

0.553

MZCO40531H11101XMR1

2 27 UPenn 1488 77 Male 17 Past 17.5 Benign Granuloma NA 0.774

MZCO43431H11101XMR1

2 28 UPenn 20069 77 Male 13 Past 12 Cancer Mixed NA 0.814

MZCO49131H11101XMR1

2 29 UPenn 20325 64 Female 9 Current 50 Cancer Adenocarcinoma NA 0.597


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MZCO41531H11101XMR1


MZCO45531H11101XMR1


MZCO46431H11101XMR1

2 32 UPenn 20183 56 Female 16 Never 0 Benign Hamartoma NA 0.334

MXCC05431H11101XMR3


MXCC05401I11101XMR1


MZCO42101I11101XMR1

3 2 UPenn 1753 48 Female 13 Past 22.5 Benign Granuloma NA 0.056

MZCO45201I11101XMR1


MZCO37001I11101XMR1

3 4 UPenn 1297 69 Female 14 Past 40 Cancer Squamous NA 0.844

MZCO52501I11101XMR1

3 5 UPenn 20519 74 Female 4 Past NA Benign Other Organizing Pneumonia 0.861

MZCO47201I11101XMR1

3 6 UPenn 20239 60 Male 13 Current 40 Benign Granuloma Necrotizing Granuloma 0.392

MZCO52901I11101XMR1

3 7 UPenn 20529 61 Male 12 Past 17.5 Cancer Other Adenocarcinoma Squamous 0.879

MZCO44501I11101XMR1


MZCO52601I11101XMR1

3 9 UPenn 20524 67 Female 12 Never 0 Benign Granuloma Necrotizing Granuloma 0.273

MZCO45701I11101XMR1

3 10 UPenn 20166 59 Male 13 Past 40.5 Benign Granuloma NA 0.204

MZCO46301I11101XMR1

3 11 UPenn 20181 72 Male 21 Past 25 Cancer Squamous NA 0.790


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MZCO36801I11101XMR1


MZCO51301I11101XMR1


MZCO41601I11101XMR1

3 14 UPenn 1627 50 Female 6 Past 20 Benign Scar NA 0.261

MZCO52001I11101XMR1

3 15 UPenn 20507 51 Female 27 Past 40 Cancer Squamous NA 0.455

MXCC05401I11101XMR2


MZCO49501I11101XMR1


MZCO36401I11101XMR1

3 18 UPenn 1257 50 Male 19 Past 30 Benign Hamartoma NA 0.807

MZCO53801I11101XMR1


MZCO49001I11101XMR1


MZCO41801I11101XMR1

3 21 UPenn 1748 66 Female 6 Past 8 Benign Other Lymphoid Aggregate 0.760

MZCO49401I11101XMR1


MZCO50901I11101XMR1


MZCO47801I11101XMR1

3 24 UPenn 20277 45 Male 11 Past 2.55 Benign Granuloma Necrotizing Granuloma 0.283

MZCO40101I11101XMR1


MZCO54401I11101XMR1



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MZCO46701I11101XMR1

3 27 UPenn 20205 85 Female 15 Never 0 Benign Granuloma NA 0.291

MZCO41401I11101XMR1

3 28 UPenn 1572 77 Female 20 NA 15 Cancer Other not specified 0.992

MZCO43601I11101XMR1


MZCO48901I11101XMR1

3 30 UPenn 20312 69 Male 33 Past 98 Benign Granuloma NA 0.486

MXCC05401I11101XMR3


MXCC05402I11101XMR4


MZCO66902I11101XMR1

4 2 NYU NYU187 64 Female 23 Past 35 Benign Granuloma Necrotizing Granuloma 0.299

MZCO60102I11101XMR1

4 3 NYU NYU16 52 Female 18 Past 30 Cancer Mixed NA 0.949

MZCO60602I11101XMR1

4 4 NYU NYU173 80 Female 12 Never 0 Benign Granuloma NA 0.378

MZCO64902I11101XMR1

4 5 NYU NYU646 79 Female 21 Past NA Cancer Mixed NA 0.704

MZCO69102I11101XMR1

4 6 NYU NYU1176 73 Male 8 Past 100 Cancer Mixed Mixed Adeno BAC 0.820

MZCO61502I11101XMR1

4 7 NYU NYU514 67 Male 10 Past 30 Benign Granuloma Necrotizing Granuloma 0.605

MZCO71202I11101XMR1

4 8 NYU NYU1096 58 Female 17 Past 10 Cancer Adenocarcinoma NA 0.579

MZCO69002I11101XMR1

4 9 NYU NYU1183 53 Female 14 Never 0 Benign Granuloma Necrotizing Granuloma 0.112

MZCO67602I11101XMR1

4 10 NYU NYU841 68 Female 15 Past 40 Benign Granuloma Necrotizing Granuloma 0.667


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MZCO61702I11101XMR1


MZCO61802I11101XMR1

4 12 NYU NYU111 60 Male 10 Past 30 Cancer BAC NA 0.833

MZCO64302I11101XMR1


MZCO66802I11101XMR1

4 14 NYU NYU1240S 47 Female 16 Past 30 Benign Granuloma NA 0.064

MZCO60202I11101XMR1

4 15 NYU NYU268 48 Female 12 Current 30 Cancer Mixed NA 0.841

MXCC05402I11101XMR5


MZCO62302I11101XMR1

4 17 NYU NYU1317S 60 Female 8 Past 40 Cancer Mixed Mixed Adeno BAC 0.896

MZCO70402I11101XMR1

4 18 NYU NYU986 37 Female 9 Never 0 Benign Other Fibrosis 0.193

MZCO66202I11101XMR1


MZCO67202I11101XMR1

4 20 NYU NYU875 54 Male 13 Past 60 Cancer Mixed NA 0.867

MZCO71402I11101XMR1

4 21 NYU NYU907 78 Female 13 Past 60 Cancer Other Pleomorphic Carcinoma 0.902

MZCO62102I11101XMR1

4 22 NYU NYU332 80 Female 13 Past 30 Benign Hamartoma NA 0.597

MZCO69302I11101XMR1

4 23 NYU NYU1145 70 Female 10 Past 3.5 Benign Other Sclerosing Hemangioma 0.313

MZCO65002I11101XMR1


MZCO63802I11101XMR1



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MZCO70202I11101XMR1

4 26 NYU NYU1001 86 Male 9 Never 0 Benign Granuloma Necrotizing Granuloma 0.770

MZCO61902I11101XMR1

4 27 NYU NYU92 51 Female 21 Never 0 Cancer Mixed NA 0.894

MZCO63102I11101XMR1

4 28 NYU NYU24 51 Female 13 Never 0 Benign Hamartoma NA 0.478

MZCO61002I11101XMR1

4 29 NYU NYU84 55 Female 19 Past NA Benign Granuloma Non-necrotizing Granuloma 0.764

MZCO63902I11101XMR1

4 30 NYU NYU697S 65 Female 8 Current 30 Cancer Mixed Mixed Adeno BAC 0.660

MXCC05402I11101XMR6A


MXCC05403I11101XMR1


MZCO69203I11101XMR1


MZCO64403I11101XMR1

5 3 NYU NYU661 79 Female 10 Never 0 Cancer Mixed Mixed Adeno BAC 0.603

MZCO61403I11101XMR1

5 4 NYU NYU513 66 Female 10 Never 0 Benign Granuloma Non-necrotizing Granuloma 0.459

MZCO65103I11101XMR1

5 5 NYU NYU613S 64 Female 10 Past 40 Cancer Mixed Mixed Adeno BAC 0.713

MZCO71103I11101XMR1


MZCO64603I11101XMR1


MZCO69703I11101XMR1

5 8 NYU NYU1098 67 Female 9 Past 80 Benign Other Organizing Pneumonia 0.859

MZCO65303I11101XMR1

5 9 NYU NYU636 67 Female 8 Past 10 Cancer BAC NA 0.928


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MZCO63403I11101XMR1


MZCO68003I11101XMR1


MZCO66303I11101XMR1

5 12 NYU NYU410 75 Male 14 Past 100 Cancer Mixed NA 0.933

MZCO62403I11101XMR1

5 13 NYU NYU281 73 Male 19 Past 70.5 Benign Granuloma NA 0.288

MZCO68203I11101XMR1

5 14 NYU NYU819 67 Female 43 Never 0 Benign Granuloma NA 0.024

MZCO61603I11101XMR1


MXCC05403I11101XMR2


MZCO68903I11101XMR1

5 17 NYU NYU1559S 66 Female 13 Past 40 Cancer Mixed NA 0.965

MZCO64703I11101XMR1


MZCO60403I11101XMR1

5 19 NYU NYU209 57 Female 21 Past 36 Benign Granuloma NA 0.406

MZCO64003I11101XMR1


MZCO62203I11101XMR1


MZCO70703I11101XMR1

5 22 NYU NYU968 72 Male 16 Past 8 Benign Granuloma NA 0.248

MZCO62503I11101XMR1

5 23 NYU NYU566 72 Female 23 Past NA Benign Granuloma NA 0.474

MZCO63703I11101XMR1

5 24 NYU NYU46 69 Female 13 Past 40 Cancer BAC NA 0.966


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MZCO67703I11101XMR1

5 25 NYU NYU830 60 Female 13 Past NA Cancer Mixed NA 0.805

MZCO66003I11101XMR1

5 27 NYU NYU440 75 Male 10 Past 7.5 Benign Granuloma Necrotizing Granuloma 0.939

MZCO61203I11101XMR1

5 28 NYU NYU560 78 Male 16 Current 120 Cancer Adenocarcinoma NA 0.943

MZCO67003I11101XMR1

5 29 NYU NYU912 64 Female 39 Never 0 Benign Other Organizing Pneumonia 0.670

MZCO71303I11101XMR1


MZCO65903I11101XMR1


MZCO63303I11101XMR1

5 32 NYU NYU65 71 Female 30 Past NA Benign Granuloma NA 0.213

MXCC05403I11101XMR3


Note:

• Sample name: Sample identifier

• Batch: Experimental batch

• Position: Sample position in the batch

• Site

o Participating study sites: IUCPQ (Institut Universitaire de Cardiologie et de Pneumologie de Quebec), NYU (New

York University), UPenn (University of Pennsylvania), and Vanderbilt (Vanderbilt University). HPS (human

plasma standard) samples are also listed.

• Subject: Unique subject identifier by study site.

• Age: Age of patient


82

• Gender: Female/Male

• Nodule size (mm): Radiological nodule size.

• Smoking status: Never/Former/Current

• Smoking pack-years: Smoking history pack-years.

• Disease status: Benign/Cancer

• Histology subgroup

o Benign – Granuloma, Hamartoma, Scar, Other.

o Cancer – Adenocarcinoma, Squamous, Large Cell, BAC, Mixed, Other.

• Histology specific: Specific histology

• Classifier score: Score by the 13-protein lung nodule classifier.


83

Table S7. List of 36 cooperative proteins.

Cat

egor

y

Pro

tein

(H

UM

AN

)

Off

icia

l gen

e na

me

Coo

pera

tive

sc

ore

Par

tial

AU

C

Coe

ffic

ient

CV

Fre

quen

cy

Tra

nsit

ion

for

quan

tita

tion

P v

alue

(M

ann-

Whi

tney

tes

t)

Tra

nsit

ion

for

qual

ific

atio

n

Pep

tide

Q

valu

e

Tis

sue

cand

idat

e

Pre

dict

ed

Con

cent

rati

on

(ng/

ml)

Classifier TSP1 THBS1 1.8 0.25 0.24 59 GFLLLASLR_495.31_559.40

0.23 GFLLLASLR_495.31_318.20

1.90E-05 510

Classifier COIA1 COL18A1 3.7 0.16 0.25 91 AVGLAGTFR_446.26_721.40

0.16 AVGLAGTFR_446.26_551.30

6.70E-04 35

Classifier ISLR ISLR 1.4 0.32 0.25 64 ALPGTPVASSQPR_640.85_841.50

0.74 ALPGTPVASSQPR_640.85_440.30

4.40E-03 -

Classifier TETN CLEC3B 2.5 0.26 0.26 67 LDTLAQEVALLK_657.39_330.20

0.14 LDTLAQEVALLK_657.39_871.50

3.70E-05 58000

Classifier FRIL FTL 2.8 0.31 0.26 53 LGGPEAGLGEYLFER_804.40_913.40

0.19 LGGPEAGLGEYLFER_804.40_525.30

4.30E-05 Secreted, EPI, ENDO

12

Classifier GRP78 HSPA5 1.4 0.27 0.27 40 TWNDPSVQQDIK_715.85_260.20

0.44 TWNDPSVQQDIK_715.85_288.10


100

Classifier ALDOA ALDOA 1.3 0.26 0.28 88 ALQASALK_401.25_617.40

0.57 ALQASALK_401.25_489.30

3.70E-05 Secreted, EPI

250

Classifier BGH3 TGFBI 1.8 0.21 0.28 69 LTLLAPLNSVFK_658.40_804.50

0.57 LTLLAPLNSVFK_658.40_875.50

1.40E-04 140

Classifier LG3BP LGALS3BP 4.3 0.29 0.29 76 VEIFYR_413.73_598.30 0.45 VEIFYR_413.73_485.30 2.80E-05 Secreted 440 Classifier LRP1 LRP1 4.0 0.13 0.32 93 TVLWPNGLSLDIPAG

R_855.00_400.20 0.26 TVLWPNGLSLDIPAGR

_855.00_605.30 1.40E-04 EPI 20

Classifier FIBA FGA 1.1 0.31 0.35 11 NSLFEYQK_514.76_714.30

0.57 NSLFEYQK_514.76_315.20

1.90E-05 130000

Classifier PRDX1 PRDX1 1.5 0.32 0.37 68 QITVNDLPVGR_606.30_428.30

0.24 QITVNDLPVGR_606.30_770.40

1.90E-05 EPI 60

Classifier GSLG1 GLG1 1.2 0.34 0.45 23 IIIQESALDYR_660.86_338.20

0.27 IIIQESALDYR_660.86_724.40

6.70E-03 EPI, ENDO

-

Robust KIT KIT 1.4 0.33 0.46 28 YVSELHLTR_373.21_263.10

0.27 YVSELHLTR_373.21_526.30

2.40E-03 8.2


84


Cat

egor

y

Pro

tein

(H

UM

AN

)

Off

icia

l gen

e na

me

Coo

pera

tive

sc

ore

Par

tial

AU

C

Coe

ffic

ient

CV

Fre

quen

cy

Tra

nsit

ion

for

quan

tita

tion

P v

alue

(M

ann-

Whi

tney

tes

t)

Tra

nsit

ion

for

qual

ific

atio

n

Pep

tide

Q

valu

e

Tis

sue

cand

idat

e

Pre

dict

ed

Con

cent

rati

on

(ng/

ml)

Robust CD14 CD14 4.0 0.33 0.48 73 ATVNPSAPR_456.80_527.30

0.72 ATVNPSAPR_456.80_386.20

4.30E-04 EPI 420

Robust EF1A1 EEF1A1 1.2 0.32 0.56 52 IGGIGTVPVGR_513.30_428.30

0.53 IGGIGTVPVGR_513.30_628.40


61

Robust TENX TNXB 1.1 0.30 0.56 22 YEVTVVSVR_526.29_759.50

0.54 YEVTVVSVR_526.29_660.40

1.10E-03 ENDO 70

Robust AIFM1 AIFM1 1.4 0.32 0.70 6 ELWFSDDPNVTK_725.85_558.30

0.20 ELWFSDDPNVTK_725.85_875.40

3.70E-02 EPI, ENDO

1.4

Robust GGH GGH 1.3 0.32 0.81 43 YYIAASYVK_539.28_638.40

0.24 YYIAASYVK_539.28_567.30

1.70E-03 250

Robust IBP3 IGFBP3 3.4 0.32 1.82 58 FLNVLSPR_473.28_685.40

0.04 FLNVLSPR_473.28_359.20

2.80E-05 5700

Robust ENPL HSP90B1 1.1 0.29 5.90 22 SGYLLPDTK_497.27_460.20

0.57 SGYLLPDTK_497.27_573.30


88

Non-Robust

ERO1A ERO1L 6.2 VLPFFERPDFQLFTGNK_685.70_318.20

0.06 VLPFFERPDFQLFTGNK_685.70_419.20


-

Non-Robust

6PGD PGD 4.3 LVPLLDTGDIIIDGGNSEYR_1080.60_897.40

0.03 LVPLLDTGDIIIDGGNSEYR_1080.60_974.50

5.50E-03 EPI, ENDO

29

Non-Robust

ICAM1 ICAM1 3.9 VELAPLPSWQPVGK_760.93_342.20

0.31 VELAPLPSWQPVGK_760.93_413.20

2.80E-02 71

Non-Robust

PTPA PPP2R4 2.1 FGSLLPIHPVTSG_662.87_807.40

0.26 FGSLLPIHPVTSG_662.87_292.10

1.90E-03 ENDO 3.3

Non-Robust

NCF4 NCF4 2.0 GATGIFPLSFVK_618.85_837.50

0.11 GATGIFPLSFVK_618.85_690.40

7.90E-04 ENDO -

Non-Robust

SEM3G SEMA3G 1.9 LFLGGLDALYSLR_719.41_837.40

0.20 LFLGGLDALYSLR_719.41_538.30

1.10E-03 -


85


Cat

egor

y

Pro

tein

(H

UM

AN

)

Off

icia

l gen

e na

me

Coo

pera

tive

sc

ore

Par

tial

AU

C

Coe

ffic

ient

CV

Fre

quen

cy

Tra

nsit

ion

for

quan

tita

tion

P v

alue

(M

ann-

Whi

tney

tes

t)

Tra

nsit

ion

for

qual

ific

atio

n

Pep

tide

Q

valu

e

Tis

sue

cand

idat

e

Pre

dict

ed

Con

cent

rati

on

(ng/

ml)

Non-Robust

1433T YWHAQ 1.5 TAFDEAIAELDTLNEDSYK_1073.00_748.40

0.69 TAFDEAIAELDTLNEDSYK_1073.00_969.50

1.10E-02 EPI 180

Non-Robust

RAP2B RAP2B 1.5 VDLEGER_409.21_603.30

0.34 VDLEGER_409.21_361.20

1.20E-03 EPI -

Non-Robust

MMP9 MMP9 1.4 AFALWSAVTPLTFTR_840.96_290.20

0.36 AFALWSAVTPLTFTR_840.96_589.30

4.00E-03 28

Non-Robust

FOLH1 FOLH1 1.3 LGSGNDFEVFFQR_758.37_825.40

0.06 LGSGNDFEVFFQR_758.37_597.30

5.80E-03 -

Non-Robust

GSTP1 GSTP1 1.3 ALPGQLKPFETLLSQNQGGK_709.39_831.40

0.46 ALPGQLKPFETLLSQNQGGK_709.39_261.20

1.70E-04 ENDO 32

Non-Robust

EF2 EEF2 1.3 FSVSPVVR_445.76_470.30

0.79 FSVSPVVR_445.76_557.30


30

Non-Robust

RAN RAN 1.2 LVLVGDGGTGK_508.29_591.30

0.27 LVLVGDGGTGK_508.29_326.20


4.6

Non-Robust

SODM SOD2 1.2 NVRPDYLK_335.52_260.20

0.86 NVRPDYLK_335.52_423.30

2.40E-02 Secreted 7.1

Non-Robust

DSG2 DSG2 1.1 GQIIGNFQAFDEDTGLPAHAR_753.04_299.20

0.08 GQIIGNFQAFDEDTGLPAHAR_753.04_551.30

5.70E-03 ENDO 2.7

Note:

• Category o Classifier: The protein belongs to the 13-protein classifier. o Robust: The protein is a cooperative protein with robust signal. o Non-Robust: The protein is a cooperative protein but with noisy signal or having interference.

• Protein (_HUMAN): UniProt protein name • Official gene name: Entrez gene symbol


86

• Cooperative score: The ratio between the occurrences of the protein in the top 100 panels and the expected occurrences by chance alone.

• Partial AUC: The partial area under the receiver operating characteristic (ROC) curve (AUC) (37, 68) of the classifier just before the protein was removed from the classifier.

• Coefficient CV: The coefficient of variation (CV) of the logistic regression coefficient just before the protein was removed from the classifier.

• Frequency: How many times the protein occurred in the top 100 panels out of all possible panels of the 21 robust cooperative proteins.

• Transition for quantitation: The transition (quantifier) used for measuring protein abundance in samples. It is the transition having the highest median intensity among all transitions of the protein.

• P value (Mann-Whitney test): The p value of the quantifier transition in distinguishing cancer vs. benign samples, based on Mann-Whitney test (31).

• Transition for qualification: The transition (together with the quantifier transition) used for calculating peptide q value. It is also the transition having the second highest median intensity among all transitions of the peptide.

• Peptide Q value: Peptide q value: See Supplementary Materials and Methods for more details. • Tissue candidate: Whether the protein was selected as a tissue biomarker candidate: See Table S3. • Predicted Concentration (ng/ml): Protein concentration in blood (69).


87

Table S8. Results from logistic regression diagnostics.

Diagnostics Results Discussion Model specification Linear predicted value: p < 0.001

Linear predicted value squared: p = 0.11

The model had relevant predictors and their linear combination was a sufficient predictor.

Goodness-of-fit

(Hosmer-Lemeshow

chi-square)

p = 0.82 The model fitted the data well.

Collinearity The lowest tolerance was 0.39 and the highest variance inflation factor (VIF) was 2.54.

No obvious collinearity was observed.

Influential observations

Standardized Pearson residual: 11 samples had an absolute value greater than 2.

Deviance residual: 2 samples had an absolute value greater than 2.

Leverage: No sample had a value greater than 3 times of the average.

Using the step-down Šidák procedure (71) to control multiple testing error (α=0.05), there were three outlier samples by the standardized Pearson residual method and none by the deviance residual method. Combining all evidence, it is inconclusive whether there were outlier samples in the dataset.

A logistic regression model was fitted to the whole discovery dataset. Technical details can be found in (35, 70).


88

Table S9. Experimental design of the validation study.

Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

ing

stat

us

Smok

ing

pack

-yea

rs

Dis

ease

st

atus

His

tolo

gy

subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

Validation Set 1 MXCC05430H11101XMR1 1 1 HPS HPS NA NA NA NA NA NA NA NA 0.247 MZCO54930H11101XMR1 1 3 IUCPQ 1842 74 Female 19 Current 50 Cancer Squamous NA 0.858 MZCO56430H11101XMR1 1 4 IUCPQ 2222 62 Male 19 Current 70.5 Cancer Squamous NA 0.915 MZCO56630H11101XMR1 1 5 IUCPQ 2324 62 Male 24 Past 50 Benign Other

Fibrotic Inflammatory Lesion 0.892

MZCO58230H11101XMR1 1 6 IUCPQ 2788 58 Female 10 Past 45 Cancer Mixed NA 0.701 MZCO57830H11101XMR1 1 7 IUCPQ 2676 58 Female 15 Past 129 Benign Scar NA 0.779 MZCO55330H11101XMR1 1 8 IUCPQ 1955 36 Female 12 Current 10.5 Benign Other Lymphangioma 0.737 MZCO57430H11101XMR1 1 9 IUCPQ 2475 64 Female 10 Past 33.75 Cancer Adenocarcinoma NA 0.144 MZCO59330H11101XMR1 1 10 IUCPQ 3076 68 Male 20 Past 40 Benign Hamartoma NA 0.085 MZCO55430H11101XMR1 1 11 IUCPQ 1958 68 Male 14 Past 56 Cancer Adenocarcinoma NA 0.437 MZCO57330H11101XMR1 1 14 IUCPQ 2450 62 Female 23 Past NA Benign Other Pulmonary Infarct 0.678 MZCO56230H11101XMR1 1 15 IUCPQ 2116 58 Female 22 Current 19 Cancer Mixed NA 0.790 MXCC05430H11101XMR2 1 16 HPS HPS NA NA NA NA NA NA NA NA 0.116 MZCO55130H11101XMR1 1 26 IUCPQ 1871 64 Female 26 Never 0 Benign Granuloma Necrotizing Granuloma 0.850 MXCC05430H 1 29 HPS HPS NA NA NA NA NA NA NA NA 0.259


89


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

ing

stat

us

Smok

ing

pack

-yea

rs

Dis

ease

st

atus

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tolo

gy

subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

11101XMR1A MXCC05431H11101XMR4 2 1 HPS HPS NA NA NA NA NA NA NA NA 0.039 MZCO46231H11101XMR1 2 2 UPenn 20177 68 Male 26 Past 78 Benign Granuloma NA 0.090 MZCO41731H11101XMR1 2 3 UPenn 1692 72 Male 15 Past 100 Cancer Squamous NA 0.863 MZCO43131H11101XMR1 2 4 UPenn 20039 57 Male 30 Past 40 Cancer Adenocarcinoma NA 0.437 MZCO38231H11101XMR1 2 5 UPenn 1399 58 Male 25 Current 40 Benign Granuloma NA 0.759 MZCO53331H11101XMR1 2 7 UPenn 20548 75 Female 14 Past 15.5 Cancer Adenocarcinoma NA 0.783 MZCO45931H11101XMR1 2 9 UPenn 20171 83 Male 14 Never 0 Benign Granuloma Necrotizing Granuloma 0.982 MZCO53731H11101XMR1 2 10 UPenn 20576 55 Male 22 Past 15 Benign Hamartoma NA 0.897 MXCC05431H11101XMR5 2 12 HPS HPS NA NA NA NA NA NA NA NA 0.106 MZCO36131H11101XMR1 2 14 UPenn 1238 59 Female 16 Past 30 Cancer Adenocarcinoma NA 0.616 MZCO44831H11101XMR1 2 16 UPenn 20144 66 Male 19 Past 45 Cancer Squamous NA 0.583 MZCO41231H11101XMR1 2 17 UPenn 1553 61 Female 19 Past 40 Cancer Adenocarcinoma NA 0.617 MZCO48031H11101XMR1 2 18 UPenn 20283 36 Female 21 Past 3.5 Benign Hamartoma NA 0.212 MZCO53931H11101XMR1 2 19 UPenn 20581 49 Male 18 Past 30 Cancer Squamous NA 0.815 MXCC05431H 2 23 HPS HPS NA NA NA NA NA NA NA NA 0.060


90


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

ing

stat

us

Smok

ing

pack

-yea

rs

Dis

ease

st

atus

His

tolo

gy

subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

11101XMR6 MXCC05401I11101XMR4 3 1 HPS HPS NA NA NA NA NA NA NA NA 0.052 MZCO62001I11101XMR1 3 2 NYU NYU330 71 Female 20 Past NA Cancer Mixed NA 0.504 MZCO67501I11101XMR1 3 3 NYU NYU862 68 Female 17 Past 52 Benign Granuloma Necrotizing Granuloma 0.480 MZCO69601I11101XMR1 3 4 NYU NYU1117 65 Male 24 Past 30 Benign Granuloma NA 0.970 MZCO68501I11101XMR1 3 6 NYU NYU777 60 Female 25 Current 90 Cancer Mixed NA 0.531 MZCO63201I11101XMR1 3 8 NYU NYU28 64 Female 13 Past NA Benign Granuloma Necrotizing Granuloma 0.615 MZCO64201I11101XMR1 3 12 NYU NYU457 74 Female 20 Never 0 Cancer Mixed NA 0.484 MZCO62601I11101XMR1 3 13 NYU NYU533 84 Female 21 Never 0 Benign Granuloma Necrotizing Granuloma 0.612 MZCO60701I11101XMR1 3 15 NYU NYU176 38 Female 22 Never 0 Benign Granuloma Necrotizing Granuloma 0.629 MXCC05401I11101XMR5 3 16 HPS HPS NA NA NA NA NA NA NA NA 0.280 MZCO70501I11101XMR1 3 17 NYU NYU974 77 Male 15 Past NA Benign Granuloma NA 0.547 MZCO60501I11101XMR1 3 18 NYU NYU293 77 Male 20 Current 160 Cancer Squamous NA 0.898 MZCO62901I11101XMR1 3 20 NYU NYU575 61 Female 27 Past 40 Cancer Adenocarcinoma NA 0.340 MZCO68101I11101XMR1 3 22 NYU NYU815 52 Female 24 Past 15 Benign Hamartoma NA 0.133 MZCO66501I1 3 25 NYU NYU409 60 Female 12 NA 60 Benign Hamartoma NA 0.086


91


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

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stat

us

Smok

ing

pack

-yea

rs

Dis

ease

st

atus

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tolo

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subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

1101XMR1A MZCO69801I11101XMR1 3 29 NYU NYU1044 52 Male 14 Current 30 Cancer Squamous NA 0.960 MZCO62801I11101XMR1 3 31 NYU NYU144 54 Female 5 Past 20 Benign Other Respiratory Bronchiolitis 0.119 MXCC05401I11101XMR6 3 33 HPS HPS NA NA NA NA NA NA NA NA 0.175 MXCC05402I11101XMR1 4 1 HPS HPS NA NA NA NA NA NA NA NA 0.090 MZCO40602I11101XMR1 4 4 UPenn 1490 74 Male 22 Never 0 Benign Other Organizing Pneumonia 0.916 MZCO51702I11101XMR1 4 6 UPenn 20488 57 Female 19 Past 18.5 Cancer Adenocarcinoma NA 0.243 MZCO39602I11101XMR1 4 7 UPenn 1450 58 Female 13 Current 40 Benign Granuloma Necrotizing Granuloma 0.940 MZCO41002I11101XMR1 4 8 UPenn 1527 58 Male 18 Past 102 Benign Granuloma Necrotizing Granuloma 0.483 MZCO41902I11101XMR1 4 9 UPenn 1750 62 Male 25 Past 75 Cancer Other Large Cell Squamous Cell 0.919 MZCO37402I11101XMR1 4 10 UPenn 1328 54 Male 24 Never 0 Benign Scar NA 0.988 MZCO36502I11101XMR1 4 11 UPenn 1266 61 Male 16 Past 60 Cancer Adenocarcinoma NA 0.779 MXCC05402I11101XMR2 4 12 HPS HPS NA NA NA NA NA NA NA NA 0.067 MZCO44102I11101XMR1 4 13 UPenn 20119 67 Female 18 Past 27 Benign Granuloma Necrotizing Granuloma 0.921 MZCO40702I11101XMR1 4 14 UPenn 1492 75 Female 10 Current 25 Cancer Squamous NA 0.372 MZCO47502I1 4 16 UPenn 20251 56 Male 26 Past 10 Benign Other Intussusception 0.042


92


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

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stat

us

Smok

ing

pack

-yea

rs

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ease

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spec

ific

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1101XMR1 MZCO46902I11101XMR1 4 17 UPenn 20222 65 Male 25 Past 43 Benign Other Lymphocytes Histiocytes 0.830 MZCO51102I11101XMR1 4 18 UPenn 20454 62 Male 23 Past 23.5 Cancer Squamous NA 0.754 MZCO39702I11101XMR1 4 21 UPenn 1452 63 Male 20 Past 20 Cancer Adenocarcinoma NA 0.872 MZCO39902I11101XMR1 4 22 UPenn 1462 65 Male 25 Past 50 Benign Granuloma Necrotizing Granuloma 0.835 MZCO50302I11101XMR1 4 23 UPenn 20434 62 Male 24 Current 92 Cancer Squamous NA 0.789 MXCC05402I11101XMR3 4 25 HPS HPS NA NA NA NA NA NA NA NA 0.023 Validation Set 2 MXCC05426E12101XMR1 1 1 HPS HPS NA NA NA NA NA NA NA NA 0.071 MZEB33326E12101XMR1 1 2

Vanderbilt 7517 74 Male 14 Past 100 Cancer Squamous NA 0.451

MZEB32226E12101XMR1 1 3

Vanderbilt 7320 71 Female 10 Current 100 Benign Other Bronchial Epithelial Cells 0.591

MZEB35726E12101XMR1 1 4

Vanderbilt 8339 62 Female 16 Never 0 Benign Granuloma Necrotizing Granuloma 0.737

MZEB32926E12101XMR1 1 5

Vanderbilt 7476 56 Female 12 Past 5 Cancer Other Carcinoid 0.598

MZEB33426E12101XMR1 1 6

Vanderbilt 7615 56 Male 8 Current 40 Cancer Squamous NA 0.520

MZEB34826E12101XMR1 1 7

Vanderbilt 7753 53 Male 12 Past 22.5 Benign Granuloma NA 0.264

MZEB18626E12101XMR1 1 9

Vanderbilt 4350 78 Female 15 Never 0 Cancer Large Cell NA 0.896


93


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

ing

stat

us

Smok

ing

pack

-yea

rs

Dis

ease

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His

tolo

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subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

MZEB24126E12101XMR1 1 10

Vanderbilt 6519 44 Female 15 Never 0 Benign Granuloma NA 0.338

MZEB19526E12101XMR1 1 11

Vanderbilt 5058 42 Female 17 Past 5 Cancer Other NSCLC Not Specified 0.909

MXCC05426E12101XMR2 1 12 HPS HPS NA NA NA NA NA NA NA NA 0.105 MZEB23526E12101XMR1 1 14

Vanderbilt 6404 73 Male 20 Past 70 Cancer Adenocarcinoma NA 0.866

MZEB32626E12101XMR1 1 15

Vanderbilt 7418 71 Male 12 Never 0 Benign Granuloma NA 0.267

MZEB26626E12101XMR1 1 16


MZEB35426E12101XMR1 1 17

Vanderbilt 7806 63 Male 10 Current 200 Benign Other Bronchial Epithelial Cells 0.752

MZEB33526E12101XMR1 1 18

Vanderbilt 7658 64 Female 15 Past 40 Benign Granuloma NA 0.767

MZEB35526E12101XMR1 1 19

Vanderbilt 7815 62 Female 11 Past 12 Cancer Adenocarcinoma NA 0.755

MZEB28426E12101XMR1 1 20

Vanderbilt 7072 68 Male 15 Past 50 Benign Other Normal 0.510

MZEB27326E12101XMR1 1 21


MZEB19026E12101XMR1 1 22


MZEB34026E12101XMR1 1 23

Vanderbilt 7680 57 Female 10 Current 30 Benign Other

Bronchiolitis Interstitial Fibrosis 0.437

MXCC05426E12101XMR4 1 24 HPS HPS NA NA NA NA NA NA NA NA 0.150 MXCC05426E12101XMR5 2 1 HPS HPS NA NA NA NA NA NA NA NA 0.032


94


Sam

ple

nam

e

Bat

ch

Pos

itio

n

Site

Subj

ect

Age

Gen

der

Nod

ule

size

(m

m)

Smok

ing

stat

us

Smok

ing

pack

-yea

rs

Dis

ease

st

atus

His

tolo

gy

subg

roup

His

tolo

gy

spec

ific

Cla

ssif

ier

scor

e

MZEB34126E12101XMR1 2 2

Vanderbilt 7696 64 Male 12 Past 52 Benign Other Bronchial Epithelial Cells 0.375

MZEB23626E12101XMR1 2 3

Vanderbilt 6427 84 Female 14 Past 45 Cancer Large Cell NA 0.610

MZEB28126E12101XMR1 2 4

Vanderbilt 7041 49 Male 20 Past 20 Benign Other Bronchial Epithelial Cells 0.293

MZEB19626E12101XMR1 2 5

Vanderbilt 5080 59 Male 20 Past 70.5 Cancer Other NSCLC Not Specified 0.602

MZEB27726E12101XMR1 2 6


MZEB27526E12101XMR1 2 7

Vanderbilt 6999 35 Female 14 Never 0 Benign Granuloma NA 0.706

MZEB26326E12101XMR1 2 8

Vanderbilt 6897 38 Male 11 Never 0 Benign Other Inflammation 0.572

MZEB30326E12101XMR1 2 9


MZEB35126E12101XMR1 2 10


MZEB35026E12101XMR1 2 11

Vanderbilt 7784 63 Male 11 Never 0 Benign Granuloma NA 0.509

MXCC05426E12101XMR6 2 12 HPS HPS NA NA NA NA NA NA NA NA 0.098 MZEB25426E12101XMR1 2 14

Vanderbilt 6830 36 Male 20 Never 0 Benign Other Emphysematous Lung 0.669

MZEB21826E12101XMR1 2 15

Vanderbilt 5193 39 Male 12 Never 0 Cancer Other Carcinoid 0.770

MZEB19426E12101XMR1 2 16


MZEB32726E12101XMR1 2 17

Vanderbilt 7450 60 Male 13 Past 50 Benign Granuloma Necrotizing Granuloma 0.719


95


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MZEB26226E12101XMR1 2 18

Vanderbilt 6885 67 Male 17 Past 90 Benign Other Squamous Metaplasia 0.871

MZEB29126E12101XMR1 2 19


MZEB23826E12101XMR1 2 20


MZEB31626E12101XMR1 2 21

Vanderbilt 7266 61 Female 6 Past 25 Benign Granuloma NA 0.857

MXCC05426E12101XMR8 2 22 HPS HPS NA NA NA NA NA NA NA NA 0.054 MXCC05413F12101XMR1 3 1 HPS HPS NA NA NA NA NA NA NA NA 0.060 MZEB58013F12101XMR1 3 2 IUCPQ 3550 60 Female 14 Past NA Cancer Adenocarcinoma NA 0.832 MZEB53813F12101XMR1 3 3 IUCPQ 4353 60 Female 11 Never 0 Benign Granuloma NA 0.400 MZEB57613F12101XMR1 3 4 IUCPQ 3516 61 Female 12 Past 9.5 Cancer Adenocarcinoma NA 0.906 MZEB57813F12101XMR1 3 5 IUCPQ 3529 56 Female 11 Current 40 Benign Other Pulmonary Infarct 0.115 MZEB59413F12101XMR1 3 6 IUCPQ 3754 69 Male 14.2 Past 30 Benign Other Amyloidosis 0.722 MZEB58813F12101XMR1 3 7 IUCPQ 3735 67 Male 19 Past 50 Cancer Adenocarcinoma NA 0.816 MZEB56613F12101XMR1 3 8 IUCPQ 3463 62 Female 22 Current 39 Cancer Adenocarcinoma NA 0.761 MZEB58413F12101XMR1 3 9 IUCPQ 3664 59 Female 24 Past 47.5 Benign Granuloma NA 0.586 MZEB55513F12101XMR1 3 10 IUCPQ 3363 58 Female 17 Current 13.8 Cancer Adenocarcinoma NA 0.158


96


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MXCC05413F12101XMR2 3 12 HPS HPS NA NA NA NA NA NA NA NA 0.111 MZEB53713F12101XMR1 3 14 IUCPQ 4229 60 Male 12 Past 40 Benign Hamartoma NA 0.943 MZEB59513F12101XMR1 3 15 IUCPQ 3755 58 Male 14 Past 40 Cancer Adenocarcinoma NA 0.793 MZEB54013F12101XMR1 3 16 IUCPQ 4454 68 Female 11 Never 0 Benign Granuloma NA 0.988 MZEB60113F12101XMR1 3 17 IUCPQ 3823 67 Female 13 Past 35 Cancer Squamous NA 0.994 MXCC05413F12101XMR6 3 24 HPS HPS NA NA NA NA NA NA NA NA 0.677

Note:

• Sample name: Sample identifier

• Batch: Experimental batch

• Position: Sample position in the batch

• Site

o Participating study sites: IUCPQ (Institut Universitaire de Cardiologie et de Pneumologie de Quebec), NYU (New

York University), UPenn (University of Pennsylvania), and Vanderbilt (Vanderbilt University). HPS (human

plasma standard) samples are also listed.

• Subject: Unique subject identifier by study site.

• Age: Age of patient

• Gender: Female/Male

• Nodule size (mm): Radiological nodule size.

• Smoking status: Never/Former/Current


97

• Smoking pack-years: Smoking history pack-years.

• Disease status: Benign/Cancer

• Histology subgroup

o Benign – Granuloma, Hamartoma, Scar, Other.

o Cancer – Adenocarcinoma, Squamous, Large Cell, BAC, Mixed, Other.

• Histology specific: Specific histology

• Classifier score: Score by the 13-protein lung nodule classifier.


98

Table S10. Impact of clinical characteristics on classifier score.

Continuous clinical characteristics Characteristics Sample

Group Pearson Correlation

Coefficient of Linear Fit

95% CI* of Coefficient

p-value on Coefficient

Subject Age All 0.190 0.005 (0.002, -0.008) 0.003 Cancer 0.015 0.000 (-0.004, -0.004) 0.871 Benign 0.227 0.005 (0.001, -0.010) 0.012 Smoking history pack-years All 0.185 0.002 (0.000, -0.003) 0.005 Cancer 0.089 0.001 (-0.001, -0.002) 0.339 Benign 0.139 0.001 (0.000, -0.003) 0.140 Nodule Size All -0.071 -0.003 (-0.008, -0.002) 0.267 Cancer -0.081 -0.003 (-0.009, -0.003) 0.368 Benign -0.035 -0.001 (-0.008, -0.005) 0.700 Categorical clinical characteristics Characteristics Classifier

Score Cancer p-value on

Cancer Benign p-value on

Benign Gender 0.477† 0.110†

Female Median (quartile range)

0.786 (0.602-0.894)

0.479 (0.282-0.721)

Male Median (quartile range)

0.815 (0.705-0.885)

0.570 (0.329-0.801)

Smoking history status

0.652‡ 0.539‡

Never Median (quartile range)

0.707 (0.558-0.841)

0.468 (0.317-0.706)

Past Median (quartile range)

0.804 (0.616-0.892)

0.510 (0.289-0.774)

Current Median (quartile range)

0.790 (0.597-0.876)

0.672 (0.437-0.759)

*Confidence interval (CI). †p-value by Mann-Whitney test. ‡ p-value by Kruskal-Wallis test.


99

Table S11. List of endogenous normalizing proteins.

Normalizing protein Transition

Median technical CV (%)

Median column drift (%)

PEDF_HUMAN LQSLFDSPDFSK_692.34_593.30 25.8 6.8 MASP1_HUMAN TGVITSPDFPNPYPK_816.92_258.10 26.5 18.3 GELS_HUMAN TASDFITK_441.73_710.40 27.1 16.8 LUM_HUMAN SLEDLQLTHNK_433.23_499.30 27.1 16.1 C163A_HUMAN INPASLDK_429.24_630.30 26.6 14.6 PTPRJ_HUMAN VITEPIPVSDLR_669.89_896.50 27.2 18.2 Normalization by panel of transitions 25.1 9.0 Without normalization 32.3 23.8

Shown are the six endogenous normalizing proteins, the corresponding transitions, and the median technical coefficients of variation (CVs) and the median column drifts before and after normalization by individual transitions and by panel of all six transitions. See also Fig. S5.


100

Table S12. Protein and peptide detection in the discovery study.

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1433E EDLVYQAK 16 4 12 0 NA NA NA 16 4 12 0 NA NA NA 1433E IISSIEQK 16 4 12 0 NA NA NA 0 0 0 0 ND ND ND 1433T AVTEQGAELSNEER 127 57 60 10 NA NA NA 0 0 0 0 ND ND ND 1433T TAFDEAIAELDTLNEDSYK 127 57 60 10 NA NA NA 127 57 60 10 0.623 4.02E-02 7.78E-03 1433Z FLIPNASQAESK 157 72 71 14 NA NA NA 157 72 71 14 0.993 5.49E-05 8.51E-05 1433Z SVTEQGAELSNEER 157 72 71 14 NA NA NA 0 0 0 0 ND ND ND 6PGD AGQAVDDFIEK 90 37 46 7 NA NA NA 0 0 0 0 ND ND ND 6PGD LVPLLDTGDIIIDGGNSEYR 90 37 46 7 NA NA NA 90 37 46 7 0.668 3.21E-02 6.59E-03 A1AG1 WFYIASAFR 157 72 70 15 NA NA NA 0 0 0 0 ND ND ND A1AG1 YVGGQEHFAHLLILR 157 72 70 15 NA NA NA 157 72 70 15 0.980 4.76E-04 3.18E-04 ABCD1 DAGIALLSITHRPSLWK 9 4 5 0 NA NA NA 0 0 0 0 ND ND ND ABCD1 GLQAPAGEPTQEASGVAA

AK 9 4 5 0 NA NA NA 0 0 0 0 ND ND ND

ABCD1 NLLTAAADAIER 9 4 5 0 NA NA NA 9 4 5 0 NA NA NA ADML LAHQIYQFTDK 27 12 15 0 NA NA NA 27 12 15 0 NA NA NA ADML SPEDSSPDAAR 27 12 15 0 NA NA NA 0 0 0 0 ND ND ND AIFM1 ELWFSDDPNVTK 158 72 71 15 NA NA NA 158 72 71 15 0.295 2.30E-01 3.64E-02 AIFM1 GVIFYLR 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND ALDOA ADDGRPFPQVIK 158 72 71 15 0.935 2.17E-03 2.48E-03 141 66 67 8 0.827 1.09E-02 2.82E-03 ALDOA ALQASALK 158 72 71 15 0.935 2.17E-03 2.48E-03 17 6 4 7 0.989 1.92E-04 1.89E-04 AMPN ALEQALEK 158 72 71 15 0.837 9.64E-03 7.21E-03 158 72 71 15 0.804 1.36E-02 3.33E-03 AMPN DHSAIPVINR 158 72 71 15 0.837 9.64E-03 7.21E-03 0 0 0 0 0.886 4.83E-03 1.47E-03 APOA1 AKPALEDLR 158 72 71 15 0.976 6.77E-04 1.78E-03 158 72 71 15 0.998 9.15E-06 2.99E-05 APOA1 ATEHLSTLSEK 158 72 71 15 0.976 6.77E-04 1.78E-03 0 0 0 0 0.998 9.15E-06 2.99E-05 APOE AATVGSLAGQPLQER 158 72 71 15 0.966 9.61E-04 1.78E-03 158 72 71 15 0.989 1.74E-04 1.89E-04 APOE LGPLVEQGR 158 72 71 15 0.966 9.61E-04 1.78E-03 0 0 0 0 0.989 1.92E-04 1.89E-04 BGH3 LTLLAPLNSVFK 158 72 71 15 0.804 1.36E-02 9.86E-03 0 0 0 0 0.976 7.05E-04 3.82E-04


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BGH3 SPYQLVLQHSR 158 72 71 15 0.804 1.36E-02 9.86E-03 158 72 71 15 0.872 6.03E-03 1.74E-03 BST1 GEGTSAHLR 157 71 71 15 NA NA NA 0 0 0 0 ND ND ND BST1 GFFADYEIPNLQK 157 71 71 15 NA NA NA 157 71 71 15 0.903 3.72E-03 1.20E-03 C163A INPASLDK 158 72 71 15 0.694 2.81E-02 1.38E-02 11 4 5 2 0.905 3.60E-03 1.19E-03 C163A LEVFYNGAWGTVGK 158 72 71 15 0.694 2.81E-02 1.38E-02 49 27 21 1 NA NA NA C163A TSYQVYSK 158 72 71 15 0.694 2.81E-02 1.38E-02 98 41 45 12 0.710 2.57E-02 5.65E-03 CALU EQFVEFR 120 59 55 6 NA NA NA 120 59 55 6 0.910 3.25E-03 1.11E-03 CALU TFDQLTPEESK 120 59 55 6 NA NA NA 0 0 0 0 ND ND ND CATB LPASFDAR 62 34 28 0 NA NA NA 62 34 28 0 0.581 4.92E-02 9.05E-03 CATB TDQYWEK 62 34 28 0 NA NA NA 0 0 0 0 ND ND ND CATG NVNPVALPR 14 4 9 1 NA NA NA 0 0 0 0 ND ND ND CATG SSGVPPEVFTR 14 4 9 1 NA NA NA 14 4 9 1 0.934 2.19E-03 8.47E-04 CBPB2 DTGTYGFLLPER 158 72 71 15 NA NA NA 158 72 71 15 0.982 3.94E-04 2.97E-04 CBPB2 EAFAAVSK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND CD14 ATVNPSAPR 158 72 71 15 0.714 2.52E-02 1.37E-02 0 0 0 0 0.932 2.28E-03 8.71E-04 CD14 SWLAELQQWLKPGLK 158 72 71 15 0.714 2.52E-02 1.37E-02 158 72 71 15 0.993 5.49E-05 8.51E-05 CD44 FAGVFHVEK 158 72 71 15 0.898 4.09E-03 3.81E-03 158 72 71 15 0.814 1.23E-02 3.10E-03 CD44 YGFIEGHVVIPR 158 72 71 15 0.898 4.09E-03 3.81E-03 0 0 0 0 0.982 4.39E-04 3.15E-04 CD59 AGLQVYNK 156 71 71 14 NA NA NA 156 71 71 14 0.867 6.49E-03 1.85E-03 CD59 TVLLLVTPFLAAAWSLHP 156 71 71 14 NA NA NA 0 0 0 0 ND ND ND CDCP1 EEGVFTVTPDTK 157 71 71 15 NA NA NA 0 0 0 0 ND ND ND CDCP1 LSLVLVPAQK 157 71 71 15 NA NA NA 157 71 71 15 0.980 4.76E-04 3.18E-04 CEAM8 LFIPNITTK 79 36 41 2 NA NA NA 79 36 41 2 NA NA NA CEAM8 TLTLLSVTR 79 36 41 2 NA NA NA 0 0 0 0 ND ND ND CERU GAYPLSIEPIGVR 158 72 71 15 0.184 3.97E-01 1.35E-01 0 0 0 0 0.876 5.57E-03 1.64E-03 CERU GPEEEHLGILGPVIWAEVG

DTIR 158 72 71 15 0.184 3.97E-01 1.35E-01 158 72 71 15 0.337 1.83E-01 2.92E-02


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CERU NNEGTYYSPNYNPQSR 158 72 71 15 0.184 3.97E-01 1.35E-01 0 0 0 0 ND ND ND CH10 GGEIQPVSVK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND CH10 VLLPEYGGTK 158 72 71 15 NA NA NA 158 72 71 15 -

0.288 9.54E-01 1.40E-01

CLIC1 FSAYIK 137 70 65 2 0.965 1.05E-03 1.78E-03 8 4 4 0 0.987 2.47E-04 2.20E-04 CLIC1 LAALNPESNTAGLDIFAK 137 70 65 2 0.965 1.05E-03 1.78E-03 129 66 61 2 0.989 1.92E-04 1.89E-04 CLIC1 NSNPALNDNLEK 137 70 65 2 0.965 1.05E-03 1.78E-03 0 0 0 0 NA NA NA CLUS ASSIIDELFQDR 158 72 71 15 0.662 3.32E-02 1.52E-02 0 0 0 0 0.935 2.16E-03 8.47E-04 CLUS EIQNAVNGVK 158 72 71 15 0.662 3.32E-02 1.52E-02 158 72 71 15 0.975 7.14E-04 3.82E-04 CNTN1 AHSDGGDGVVSQVK 158 72 71 15 0.482 8.00E-02 3.11E-02 157 72 70 15 0.679 3.03E-02 6.33E-03 CNTN1 DGEYVVEVR 158 72 71 15 0.482 8.00E-02 3.11E-02 1 0 1 0 0.482 8.01E-02 1.39E-02 CO6A3 IGDLHPQIVNLLK 158 72 71 15 0.743 2.13E-02 1.29E-02 0 0 0 0 0.598 4.55E-02 8.47E-03 CO6A3 VAVVQYSDR 158 72 71 15 0.743 2.13E-02 1.29E-02 158 72 71 15 0.823 1.12E-02 2.85E-03 CO6A3 WYYDPNTK 158 72 71 15 0.743 2.13E-02 1.29E-02 0 0 0 0 ND ND ND COF1 EILVGDVGQTVDDPYATF

VK 127 65 60 2 NA NA NA 0 0 0 0 ND ND ND

COF1 LGGSAVISLEGKPL 127 65 60 2 NA NA NA 0 0 0 0 ND ND ND COF1 YALYDATYETK 127 65 60 2 NA NA NA 127 65 60 2 0.963 1.11E-03 5.09E-04 COIA1 AVGLAGTFR 158 72 71 15 0.616 4.18E-02 1.80E-02 37 16 13 8 0.909 3.35E-03 1.12E-03 COIA1 TEAPSATGQASSLLGGR 158 72 71 15 0.616 4.18E-02 1.80E-02 121 56 58 7 0.440 1.01E-01 1.69E-02 CRP APLTKPLK 153 70 68 15 0.991 1.19E-04 1.09E-03 21 13 7 1 0.994 5.49E-05 8.51E-05 CRP ESDTSYVSLK 153 70 68 15 0.991 1.19E-04 1.09E-03 132 57 61 14 0.999 9.15E-06 2.99E-05 CRP YEVQGEVFTKPQLWP 153 70 68 15 0.991 1.19E-04 1.09E-03 0 0 0 0 ND ND ND CSF1 FNSVPLTDTGHER 134 64 59 11 -

0.117 8.66E-01 2.62E-01 113 56 50 7 0.631 3.86E-02 7.66E-03

CSF1 ISSLRPQGLSNPSTLSAQPQLSR

134 64 59 11 -0.117

8.66E-01 2.62E-01 21 8 9 4 NA NA NA


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CYTB SQLEEK 100 50 48 2 NA NA NA 0 0 0 0 ND ND ND CYTB SQVVAGTNYFIK 100 50 48 2 NA NA NA 100 50 48 2 0.924 2.59E-03 9.41E-04 DESP YGDGIQLTR 131 59 60 12 NA NA NA 131 59 60 12 NA NA NA DMKN QVPGFGVADALGNR 128 57 56 15 NA NA NA 0 0 0 0 ND ND ND DMKN VSEALGQGTR 128 57 56 15 NA NA NA 128 57 56 15 0.608 4.32E-02 8.21E-03 DSG2 GQIIGNFQAFDEDTGLPAH

AR 158 72 71 15 0.096 5.55E-01 1.78E-01 1 0 0 1 0.628 3.92E-02 7.68E-03

DSG2 ILDVNDNIPVVENK 158 72 71 15 0.096 5.55E-01 1.78E-01 157 72 71 14 0.039 6.57E-01 9.82E-02 EF1A1 IGGIGTVPVGR 158 72 71 15 -

0.004 7.28E-01 2.31E-01 158 72 71 15 0.930 2.35E-03 8.80E-04

EF1A1 QTVAVGVIK 158 72 71 15 -0.004

7.28E-01 2.31E-01 0 0 0 0 0.309 2.15E-01 3.41E-02

EF2 FSVSPVVR 125 61 55 9 NA NA NA 125 61 55 9 0.573 5.14E-02 9.33E-03 EF2 GVQYLNEIK 125 61 55 9 NA NA NA 0 0 0 0 ND ND ND ENOA AVEHINK 156 72 70 14 NA NA NA 0 0 0 0 ND ND ND ENOA YISPDQLADLYK 156 72 70 14 NA NA NA 156 72 70 14 0.802 1.37E-02 3.33E-03 ENOA YNQLLR 156 72 70 14 NA NA NA 0 0 0 0 ND ND ND ENPL SGTSEFLNK 158 72 71 15 0.735 2.23E-02 1.29E-02 1 0 1 0 0.704 2.67E-02 5.74E-03 ENPL SGYLLPDTK 158 72 71 15 0.735 2.23E-02 1.29E-02 157 72 70 15 0.904 3.64E-03 1.19E-03 EPHB6 RPHFDQLVAAFDK 157 72 70 15 NA NA NA 0 0 0 0 ND ND ND EPHB6 WAAPEVIAHGK 157 72 70 15 NA NA NA 157 72 70 15 -

0.078 8.29E-01 1.23E-01

ERBB3 GESIEPLDPSEK 105 44 49 12 NA NA NA 0 0 0 0 ND ND ND ERBB3 LAEVPDLLEK 105 44 49 12 NA NA NA 105 44 49 12 0.042 6.52E-01 9.79E-02 EREG VAQVSITK 115 58 56 1 NA NA NA 115 58 56 1 0.531 6.24E-02 1.10E-02 EREG VTSGDPELPQV 115 58 56 1 NA NA NA 0 0 0 0 ND ND ND ERO1A AVLQWTK 121 57 52 12 NA NA NA 0 0 0 0 ND ND ND


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ERO1A LLESDYFR 121 57 52 12 NA NA NA 0 0 0 0 ND ND ND ERO1A NLLQNIH 121 57 52 12 NA NA NA 0 0 0 0 ND ND ND ERO1A VLPFFERPDFQLFTGNK 121 57 52 12 NA NA NA 121 57 52 12 0.467 8.69E-02 1.48E-02 F10A1 AIDLFTDAIK 35 15 20 0 NA NA NA 0 0 0 0 ND ND ND F10A1 LQKPNAAIR 35 15 20 0 NA NA NA 35 15 20 0 NA NA NA FAM3C GINVALANGK 97 51 37 9 0.554 5.63E-02 2.32E-02 88 46 34 8 0.618 4.13E-02 7.95E-03 FAM3C SALDTAAR 97 51 37 9 0.554 5.63E-02 2.32E-02 9 5 3 1 -

0.769 9.99E-01 1.45E-01

FAM3C TGEVLDTK 97 51 37 9 0.554 5.63E-02 2.32E-02 0 0 0 0 ND ND ND FCGR1 HLEEELK 39 15 22 2 NA NA NA 0 0 0 0 ND ND ND FCGR1 VFTEGEPLALR 39 15 22 2 NA NA NA 39 15 22 2 NA NA NA FIBA GGSTSYGTGSETESPR 147 67 65 15 0.965 1.02E-03 1.78E-03 108 47 46 15 0.998 9.15E-06 2.99E-05 FIBA NSLFEYQK 147 67 65 15 0.965 1.02E-03 1.78E-03 39 20 19 0 0.999 9.15E-06 2.99E-05 FINC SYTITGLQPGTDYK 154 70 69 15 0.600 4.50E-02 1.90E-02 135 61 59 15 0.982 4.39E-04 3.15E-04 FINC VPGTSTSATLTGLTR 154 70 69 15 0.600 4.50E-02 1.90E-02 19 9 10 0 0.695 2.79E-02 5.86E-03 FKB11 ANYWLK 23 8 12 3 NA NA NA 0 0 0 0 ND ND ND FKB11 DPLVIELGQK 23 8 12 3 NA NA NA 23 8 12 3 NA NA NA FOLH1 GVILYSDPADYFAPGVK 138 62 62 14 NA NA NA 0 0 0 0 ND ND ND FOLH1 LGSGNDFEVFFQR 138 62 62 14 NA NA NA 138 62 62 14 0.706 2.65E-02 5.74E-03 FRIL DDVALEGVSHFFR 151 71 70 10 NA NA NA 0 0 0 0 ND ND ND FRIL LGGPEAGLGEYLFER 151 71 70 10 NA NA NA 151 71 70 10 0.980 5.13E-04 3.35E-04 G3P GALQNIIPASTGAAK 150 72 70 8 0.936 2.11E-03 2.48E-03 149 72 69 8 0.965 1.05E-03 4.92E-04 G3P LISWYDNEFGYSNR 150 72 70 8 0.936 2.11E-03 2.48E-03 1 0 1 0 0.958 1.29E-03 5.75E-04 G6PD DGLLPENTFIVGYAR 43 19 24 0 NA NA NA 43 19 24 0 0.995 4.58E-05 8.51E-05 G6PD GGYFDEFGIIR 43 19 24 0 NA NA NA 0 0 0 0 ND ND ND G6PI AVLHVALR 39 15 22 2 0.951 1.60E-03 2.10E-03 6 4 2 0 0.758 1.95E-02 4.47E-03 G6PI TLAQLNPESSLFIIASK 39 15 22 2 0.951 1.60E-03 2.10E-03 33 11 20 2 0.865 6.77E-03 1.91E-03


105


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GDIR2 DIVSGLK 158 72 71 15 0.151 4.54E-01 1.50E-01 158 72 71 15 0.753 2.01E-02 4.58E-03 GDIR2 LNYKPPPQK 158 72 71 15 0.151 4.54E-01 1.50E-01 0 0 0 0 0.929 2.36E-03 8.80E-04 GELS AQPVQVAEGSEPDGFWEA

LGGK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND

GELS TASDFITK 158 72 71 15 NA NA NA 158 72 71 15 0.981 4.67E-04 3.18E-04 GGH NLDGISHAPNAVK 158 72 71 15 0.772 1.75E-02 1.19E-02 158 72 71 15 0.795 1.48E-02 3.48E-03 GGH YYIAASYVK 158 72 71 15 0.772 1.75E-02 1.19E-02 0 0 0 0 0.845 8.66E-03 2.34E-03 GRP78 TWNDPSVQQDIK 158 72 71 15 0.477 8.22E-02 3.13E-02 90 42 44 4 0.843 8.91E-03 2.38E-03 GRP78 VYEGERPLTK 158 72 71 15 0.477 8.22E-02 3.13E-02 68 30 27 11 0.260 2.78E-01 4.35E-02 GSLG1 IIIQESALDYR 158 72 71 15 NA NA NA 158 72 71 15 0.574 5.11E-02 9.33E-03 GSLG1 LDPALQDK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND GSLG1 LIAQDYK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND GSLG1 NDINILK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND GSTP1 ALPGQLKPFETLLSQNQG

GK 123 61 61 1 NA NA NA 123 61 61 1 0.973 7.87E-04 4.13E-04

GSTP1 YISLIYTNYEAGK 123 61 61 1 NA NA NA 0 0 0 0 ND ND ND HPSE LPYPFSNK 49 22 24 3 NA NA NA 0 0 0 0 ND ND ND HPSE SVQLNGLTLK 49 22 24 3 NA NA NA 49 22 24 3 NA NA NA HPT VGYVSGWGR 158 72 71 15 0.994 5.49E-05 1.09E-03 0 0 0 0 0.997 9.15E-06 2.99E-05 HPT VTSIQDWVQK 158 72 71 15 0.994 5.49E-05 1.09E-03 158 72 71 15 1.000 9.15E-06 2.99E-05 HS90A SLTNDWEDHLAVK 32 16 15 1 NA NA NA 32 16 15 1 -

0.483 9.87E-01 1.44E-01

HS90B ADHGEPIGR 121 58 61 2 NA NA NA 0 0 0 0 ND ND ND HS90B IDIIPNPQER 121 58 61 2 NA NA NA 121 58 61 2 0.945 1.72E-03 7.13E-04 HS90B NPDDITQEEYGEFYK 121 58 61 2 NA NA NA 0 0 0 0 ND ND ND HSPB1 DGVVEITGK 30 14 14 2 NA NA NA 0 0 0 0 ND ND ND HSPB1 GPSWDPFR 30 14 14 2 NA NA NA 30 14 14 2 NA NA NA


106


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HTRA1 LHRPPVIVLQR 40 13 18 9 NA NA NA 40 13 18 9 0.671 3.16E-02 6.55E-03 HTRA1 LPVLLLGR 40 13 18 9 NA NA NA 0 0 0 0 ND ND ND HTRA1 VTAGISFAIPSDK 40 13 18 9 NA NA NA 0 0 0 0 ND ND ND HXK1 FLLSESGSGK 117 52 55 10 -

0.045 7.88E-01 2.41E-01 17 7 6 4 NA NA NA

HXK1 LVDEYSLNAGK 117 52 55 10 -0.045

7.88E-01 2.41E-01 47 24 21 2 NA NA NA

HXK1 SANLVAATLGAILNR 117 52 55 10 -0.045

7.88E-01 2.41E-01 53 21 28 4 NA NA NA

HYOU1 FPEHELTFDPQR 156 72 71 13 0.326 1.95E-01 7.05E-02 0 0 0 0 0.468 8.62E-02 1.47E-02 HYOU1 LPATEKPVLLSK 156 72 71 13 0.326 1.95E-01 7.05E-02 156 72 71 13 0.545 5.86E-02 1.04E-02 IBP2 AEVLFR 158 72 71 15 0.948 1.66E-03 2.10E-03 0 0 0 0 0.881 5.25E-03 1.58E-03 IBP2 ELAVFR 158 72 71 15 0.948 1.66E-03 2.10E-03 158 72 71 15 0.985 2.84E-04 2.39E-04 IBP2 LIQGAPTIR 158 72 71 15 0.948 1.66E-03 2.10E-03 0 0 0 0 0.970 8.70E-04 4.26E-04 IBP3 FHPLHSK 158 72 71 15 0.915 3.01E-03 2.92E-03 0 0 0 0 0.976 7.14E-04 3.82E-04 IBP3 FLNVLSPR 158 72 71 15 0.915 3.01E-03 2.92E-03 0 0 0 0 0.993 5.49E-05 8.51E-05 IBP3 YGQPLPGYTTK 158 72 71 15 0.915 3.01E-03 2.92E-03 158 72 71 15 0.448 9.70E-02 1.63E-02 ICAM1 ASVSVTAEDEGTQR 114 50 57 7 NA NA NA 0 0 0 0 ND ND ND ICAM1 VELAPLPSWQPVGK 114 50 57 7 NA NA NA 114 50 57 7 0.293 2.34E-01 3.68E-02 ICAM3 IALETSLSK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND ICAM3 TFVLPVTPPR 158 72 71 15 NA NA NA 158 72 71 15 0.923 2.67E-03 9.59E-04 IF4A1 GYDVIAQAQSGTGK 58 26 32 0 NA NA NA 0 0 0 0 ND ND ND IF4A1 VLITTDLLAR 58 26 32 0 NA NA NA 58 26 32 0 NA NA NA IGF1 EGTEASLQIR 40 24 16 0 NA NA NA 0 0 0 0 ND ND ND IGF1 ISSLPTQLFK 40 24 16 0 NA NA NA 40 24 16 0 NA NA NA IL18 SDIIFFQR 45 21 24 0 NA NA NA 45 21 24 0 0.594 4.63E-02 8.57E-03 IL18 SVPGHDNK 45 21 24 0 NA NA NA 0 0 0 0 ND ND ND


107


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ILEU EATTNAPFR 88 44 44 0 0.989 1.92E-04 1.25E-03 13 5 8 0 0.993 6.41E-05 8.98E-05 ILEU TYNFLPEFLVSTQK 88 44 44 0 0.989 1.92E-04 1.25E-03 75 39 36 0 0.995 4.58E-05 8.51E-05 ILK HSGIDFK 90 40 45 5 0.887 4.78E-03 4.04E-03 15 9 6 0 0.803 1.36E-02 3.33E-03 ILK QLNFLTK 90 40 45 5 0.887 4.78E-03 4.04E-03 75 31 39 5 0.917 2.97E-03 1.04E-03 ILK WQGNDIVVK 90 40 45 5 0.887 4.78E-03 4.04E-03 0 0 0 0 ND ND ND INHBA AEVWLFLK 32 16 14 2 NA NA NA 0 0 0 0 ND ND ND INHBA EGSDLSVVER 32 16 14 2 NA NA NA 32 16 14 2 -

0.573 9.93E-01 1.45E-01

ISLR ALPGTPVASSQPR 158 72 71 15 0.736 2.22E-02 1.29E-02 0 0 0 0 0.724 2.37E-02 5.24E-03 ISLR EVPLLQSLWLAHNEIR 158 72 71 15 0.736 2.22E-02 1.29E-02 0 0 0 0 0.838 9.46E-03 2.51E-03 ISLR LPGLPEGAFR 158 72 71 15 0.736 2.22E-02 1.29E-02 158 72 71 15 0.835 9.84E-03 2.59E-03 ITA5 SLQWFGATVR 114 55 53 6 NA NA NA 114 55 53 6 0.436 1.03E-01 1.72E-02 ITA5 SSASSGPQILK 114 55 53 6 NA NA NA 0 0 0 0 ND ND ND K1C18 LASYLDR 72 37 31 4 NA NA NA 0 0 0 0 ND ND ND K1C18 LQLETEIEALK 72 37 31 4 NA NA NA 72 37 31 4 NA NA NA K1C18 VVSETNDTK 72 37 31 4 NA NA NA 0 0 0 0 ND ND ND K1C19 FGAQLAHIQALISGIEAQL

GDVR 158 72 71 15 NA NA NA 158 72 71 15 0.399 1.28E-01 2.10E-02

K1C19 FGPGVAFR 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND KIT QATLTISSAR 158 72 71 15 0.481 8.03E-02 3.11E-02 158 72 71 15 0.700 2.72E-02 5.76E-03 KIT YVSELHLTR 158 72 71 15 0.481 8.03E-02 3.11E-02 0 0 0 0 0.811 1.26E-02 3.15E-03 KLK14 VLGSGTWPSAPK 27 17 10 0 NA NA NA 27 17 10 0 NA NA NA KLK14 VSGWGTISSPIAR 27 17 10 0 NA NA NA 0 0 0 0 ND ND ND KPYM APIIAVTR 158 72 71 15 0.965 1.04E-03 1.78E-03 13 3 3 7 0.993 6.41E-05 8.98E-05 KPYM LDIDSPPITAR 158 72 71 15 0.965 1.04E-03 1.78E-03 145 69 68 8 0.952 1.58E-03 6.75E-04 LAMB2 IQGTLQPHAR 69 34 35 0 -

0.348 9.68E-01 2.83E-01 0 0 0 0 ND ND ND


108


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LAMB2 SLADVDAILAR 69 34 35 0 -0.348

9.68E-01 2.83E-01 31 14 17 0 NA NA NA

LAMB2 VLELSIPASAEQIQHLAGAIAER

69 34 35 0 -0.348

9.68E-01 2.83E-01 38 20 18 0 NA NA NA

LDHA FIIPNVVK 157 72 71 14 0.890 4.58E-03 4.04E-03 0 0 0 0 0.942 1.82E-03 7.34E-04 LDHA LVIITAGAR 157 72 71 14 0.890 4.58E-03 4.04E-03 157 72 71 14 0.943 1.81E-03 7.34E-04 LDHB FIIPQIVK 158 72 71 15 0.759 1.93E-02 1.28E-02 157 72 71 14 0.954 1.53E-03 6.61E-04 LDHB GLTSVINQK 158 72 71 15 0.759 1.93E-02 1.28E-02 1 0 0 1 0.889 4.68E-03 1.45E-03 LEG1 GEVAPDAK 146 68 70 8 NA NA NA 0 0 0 0 ND ND ND LEG1 LPDGYEFK 146 68 70 8 NA NA NA 0 0 0 0 ND ND ND LEG1 SFVLNLGK 146 68 70 8 NA NA NA 146 68 70 8 0.702 2.69E-02 5.74E-03 LG3BP ASHEEVEGLVEK 158 72 71 15 0.962 1.16E-03 1.78E-03 158 72 71 15 0.984 3.84E-04 2.97E-04 LG3BP VEIFYR 158 72 71 15 0.962 1.16E-03 1.78E-03 0 0 0 0 0.990 1.19E-04 1.40E-04 LG3BP YSSDYFQAPSDYR 158 72 71 15 0.962 1.16E-03 1.78E-03 0 0 0 0 0.986 2.47E-04 2.20E-04 LRP1 TVLWPNGLSLDIPAGR 158 72 71 15 -

0.282 9.53E-01 2.83E-01 158 72 71 15 0.976 6.96E-04 3.82E-04

LRP1 VFFTDYGQIPK 158 72 71 15 -0.282

9.53E-01 2.83E-01 0 0 0 0 0.064 6.13E-01 9.25E-02

LUM NIPTVNENLENYYLEVNQLEK

158 72 71 15 0.917 2.95E-03 2.92E-03 158 72 71 15 0.978 5.77E-04 3.53E-04

LUM SLEDLQLTHNK 158 72 71 15 0.917 2.95E-03 2.92E-03 0 0 0 0 0.976 7.14E-04 3.82E-04 LYOX HWFQAGYSTSR 121 54 53 14 NA NA NA 0 0 0 0 ND ND ND LYOX TPILLIR 121 54 53 14 NA NA NA 121 54 53 14 0.599 4.53E-02 8.47E-03 MASP1 APGELEHGLITFSTR 158 72 71 15 0.457 9.16E-02 3.43E-02 151 68 70 13 0.796 1.46E-02 3.47E-03 MASP1 TGVITSPDFPNPYPK 158 72 71 15 0.457 9.16E-02 3.43E-02 7 4 1 2 0.958 1.31E-03 5.75E-04 MDHC LGVTANDVK 130 63 62 5 NA NA NA 130 63 62 5 0.547 5.79E-02 1.03E-02 MDHC VLVTGAAGQIAYSLLYSIG 130 63 62 5 NA NA NA 0 0 0 0 ND ND ND


109


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NGSVFGK MDHM VDFPQDQLTALTGR 158 72 71 15 NA NA NA 158 72 71 15 0.557 5.54E-02 1.00E-02 MDHM VSSFEEK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND MMP12 FLLILLLQATASGALPLNSS

TSLEK 158 72 71 15 NA NA NA 158 72 71 15 0.822 1.13E-02 2.87E-03

MMP12 GIQSLYGDPK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND MMP12 IDAVFYSK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND MMP2 AFQVWSDVTPLR 153 72 71 10 0.666 3.24E-02 1.52E-02 152 72 71 9 0.911 3.22E-03 1.11E-03 MMP2 IIGYTPDLDPETVDDAFAR 153 72 71 10 0.666 3.24E-02 1.52E-02 1 0 0 1 0.659 3.36E-02 6.82E-03 MMP7 LSQDDIK 102 49 47 6 NA NA NA 102 49 47 6 0.452 9.46E-02 1.60E-02 MMP7 NANSLEAK 102 49 47 6 NA NA NA 0 0 0 0 ND ND ND MMP9 AFALWSAVTPLTFTR 158 72 71 15 0.850 8.18E-03 6.32E-03 50 15 20 15 0.749 2.06E-02 4.62E-03 MMP9 FQTFEGDLK 158 72 71 15 0.850 8.18E-03 6.32E-03 108 57 51 0 0.826 1.09E-02 2.82E-03 MMP9 SLGPALLLLQK 158 72 71 15 0.850 8.18E-03 6.32E-03 0 0 0 0 0.970 8.88E-04 4.28E-04 MPRI GHQAFDVGQPR 158 72 71 15 0.203 3.64E-01 1.27E-01 23 11 9 3 0.384 1.40E-01 2.28E-02 MPRI TYHSVGDSVLR 158 72 71 15 0.203 3.64E-01 1.27E-01 4 3 0 1 0.375 1.47E-01 2.40E-02 MPRI VPIDGPPIDIGR 158 72 71 15 0.203 3.64E-01 1.27E-01 131 58 62 11 0.350 1.71E-01 2.75E-02 NCF4 AEALFDFTGNSK 138 62 61 15 0.695 2.80E-02 1.38E-02 43 18 21 4 NA NA NA NCF4 DAEGDLVR 138 62 61 15 0.695 2.80E-02 1.38E-02 0 0 0 0 ND ND ND NCF4 DIAVEEDLSSTPLLK 138 62 61 15 0.695 2.80E-02 1.38E-02 0 0 0 0 ND ND ND NCF4 GATGIFPLSFVK 138 62 61 15 0.695 2.80E-02 1.38E-02 95 44 40 11 0.972 8.15E-04 4.13E-04 NDKB DRPFFPGLVK 24 11 13 0 NA NA NA 0 0 0 0 ND ND ND NDKB NIIHGSDSVK 24 11 13 0 NA NA NA 24 11 13 0 0.419 1.14E-01 1.88E-02 NRP1 FVSDYETHGAGFSIR 158 72 71 15 0.718 2.45E-02 1.37E-02 0 0 0 0 0.481 8.03E-02 1.39E-02 NRP1 FVTAVGTQGAISK 158 72 71 15 0.718 2.45E-02 1.37E-02 158 72 71 15 0.660 3.36E-02 6.82E-03 NRP1 SFEGNNNYDTPELR 158 72 71 15 0.718 2.45E-02 1.37E-02 0 0 0 0 0.750 2.05E-02 4.62E-03 OSTP AIPVAQDLNAPSDWDSR 108 51 51 6 NA NA NA 108 51 51 6 0.603 4.44E-02 8.37E-03


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OSTP DSYETSQLDDQSAETHSHK

108 51 51 6 NA NA NA 0 0 0 0 ND ND ND

OSTP YPDAVATWLNPDPSQK 108 51 51 6 NA NA NA 0 0 0 0 ND ND ND PCBP2 IANPVEGSTDR 52 27 23 2 NA NA NA 0 0 0 0 NA NA NA PCBP2 IITLAGPTNAIFK 52 27 23 2 NA NA NA 52 27 23 2 0.169 4.23E-01 6.56E-02 PCYOX IAIIGAGIGGTSAAYYLR 37 19 16 2 NA NA NA 0 0 0 0 ND ND ND PCYOX IFSQETLTK 37 19 16 2 NA NA NA 37 19 16 2 NA NA NA PCYOX TLLETLQK 37 19 16 2 NA NA NA 0 0 0 0 ND ND ND PDGFB SFDDLQR 111 57 51 3 0.212 3.49E-01 1.24E-01 99 48 48 3 0.080 5.83E-01 8.84E-02 PDGFB SHSGGELESLAR 111 57 51 3 0.212 3.49E-01 1.24E-01 12 9 3 0 0.109 5.31E-01 8.14E-02 PDIA3 ELSDFISYLQR 129 62 59 8 NA NA NA 129 62 59 8 0.894 4.27E-03 1.35E-03 PDIA3 SEPIPESNDGPVK 129 62 59 8 NA NA NA 0 0 0 0 ND ND ND PDIA4 FDVSGYPTIK 81 35 42 4 NA NA NA 81 35 42 4 0.481 8.05E-02 1.39E-02 PDIA4 FHHTFSTEIAK 81 35 42 4 NA NA NA 0 0 0 0 ND ND ND PECA1 SELVTVTESFSTPK 77 39 34 4 NA NA NA 0 0 0 0 ND ND ND PECA1 STESYFIPEVR 77 39 34 4 NA NA NA 77 39 34 4 NA NA NA PEDF LQSLFDSPDFSK 158 72 71 15 0.961 1.21E-03 1.78E-03 0 0 0 0 0.990 1.19E-04 1.40E-04 PEDF TVQAVLTVPK 158 72 71 15 0.961 1.21E-03 1.78E-03 158 72 71 15 0.978 5.77E-04 3.53E-04 PGAM1 HGESAWNLENR 14 7 6 1 NA NA NA 14 7 6 1 NA NA NA PLIN2 DAVTTTVTGAK 138 63 61 14 NA NA NA 0 0 0 0 ND ND ND PLIN2 EVSDSLLTSSK 138 63 61 14 NA NA NA 138 63 61 14 0.083 5.79E-01 8.82E-02 PLSL IGNFSTDIK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND PLSL ISFDEFIK 158 72 71 15 NA NA NA 158 72 71 15 0.975 7.14E-04 3.82E-04 PLXB3 ELPVPIYVTQGEAQR 77 40 35 2 NA NA NA 0 0 0 0 ND ND ND PLXB3 GPVDAVTGK 77 40 35 2 NA NA NA 77 40 35 2 NA NA NA PLXC1 FWVNILK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND PLXC1 LNTIGHYEISNGSTIK 158 72 71 15 NA NA NA 158 72 71 15 NA NA NA


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POSTN GFEPGVTNILK 158 72 71 15 0.624 3.99E-02 1.75E-02 158 72 71 15 0.935 2.16E-03 8.47E-04 POSTN IIDGVPVEITEK 158 72 71 15 0.624 3.99E-02 1.75E-02 0 0 0 0 0.801 1.40E-02 3.37E-03 POSTN IIHGNQIATNGVVHVIDR 158 72 71 15 0.624 3.99E-02 1.75E-02 0 0 0 0 ND ND ND PPIB VIFGLFGK 158 72 71 15 0.926 2.47E-03 2.69E-03 0 0 0 0 0.863 6.89E-03 1.93E-03 PPIB VYFDLR 158 72 71 15 0.926 2.47E-03 2.69E-03 158 72 71 15 0.888 4.73E-03 1.45E-03 PRDX1 IGHPAPNFK 158 72 71 15 0.860 7.15E-03 5.83E-03 116 57 59 0 0.550 5.74E-02 1.03E-02 PRDX1 QITVNDLPVGR 158 72 71 15 0.860 7.15E-03 5.83E-03 42 15 12 15 0.996 1.83E-05 4.49E-05 PROF1 STGGAPTFNVTVTK 158 72 71 15 0.984 3.57E-04 1.78E-03 157 71 71 15 0.991 9.15E-05 1.22E-04 PROF1 TFVNITPAEVGVLVGK 158 72 71 15 0.984 3.57E-04 1.78E-03 1 1 0 0 0.996 1.83E-05 4.49E-05 PRS6A VDILDPALLR 13 7 5 1 NA NA NA 13 7 5 1 0.986 2.56E-04 2.22E-04 PTGIS DPEIYTDPEVFK 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND PTGIS LLLFPFLSPQR 158 72 71 15 NA NA NA 158 72 71 15 0.159 4.39E-01 6.76E-02 PTPA FGSLLPIHPVTSG 103 55 47 1 NA NA NA 103 55 47 1 0.741 2.15E-02 4.80E-03 PTPA TGPFAEHSNQLWNISAVPS

WSK 103 55 47 1 NA NA NA 0 0 0 0 ND ND ND

PTPA VDDQIAIVFK 103 55 47 1 NA NA NA 0 0 0 0 ND ND ND PTPA WIDETPPVDQPSR 103 55 47 1 NA NA NA 0 0 0 0 ND ND ND PTPRJ AVSISPTNVILTWK 158 72 71 15 0.679 3.04E-02 1.46E-02 0 0 0 0 0.916 2.98E-03 1.04E-03 PTPRJ VITEPIPVSDLR 158 72 71 15 0.679 3.04E-02 1.46E-02 158 72 71 15 0.890 4.60E-03 1.44E-03 PVR SVDIWLR 158 72 71 15 0.659 3.37E-02 1.52E-02 158 72 71 15 0.948 1.66E-03 6.96E-04 PVR VLAKPQNTAEVQK 158 72 71 15 0.659 3.37E-02 1.52E-02 0 0 0 0 0.791 1.52E-02 3.54E-03 RAB32 VHLPNGSPIPAVLLANK 22 1 6 15 NA NA NA 0 0 0 0 ND ND ND RAB32 VLVIGELGVGK 22 1 6 15 NA NA NA 22 1 6 15 NA NA NA RAN FNVWDTAGQEK 116 58 58 0 0.491 7.62E-02 3.05E-02 2 1 1 0 NA NA NA RAN LVLVGDGGTGK 116 58 58 0 0.491 7.62E-02 3.05E-02 114 57 57 0 0.637 3.75E-02 7.51E-03 RAN NVPNWHR 116 58 58 0 0.491 7.62E-02 3.05E-02 0 0 0 0 ND ND ND RAP2B EVSYGEGK 145 66 64 15 NA NA NA 0 0 0 0 ND ND ND


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RAP2B VDLEGER 145 66 64 15 NA NA NA 145 66 64 15 NA NA NA S10A1 DVDAVDK 128 56 60 12 NA NA NA 128 56 60 12 NA NA NA S10A1 ELLQTELSGFLDAQK 128 56 60 12 NA NA NA 0 0 0 0 ND ND ND S10A6 ELTIGSK 154 70 71 13 0.974 7.42E-04 1.78E-03 154 70 71 13 0.978 5.77E-04 3.53E-04 S10A6 LQDAEIAR 154 70 71 13 0.974 7.42E-04 1.78E-03 0 0 0 0 0.989 1.92E-04 1.89E-04 SAA EANYIGSDK 143 65 63 15 NA NA NA 0 0 0 0 ND ND ND SAA SFFSFLGEAFDGAR 143 65 63 15 NA NA NA 143 65 63 15 1.000 9.15E-06 2.99E-05 SCF LFTPEEFFR 143 65 65 13 NA NA NA 143 65 65 13 0.630 3.88E-02 7.66E-03 SCF LVANLPK 143 65 65 13 NA NA NA 0 0 0 0 ND ND ND SEM3G DYPDEVLQFAR 155 70 71 14 NA NA NA 0 0 0 0 ND ND ND SEM3G LFLGGLDALYSLR 155 70 71 14 NA NA NA 155 70 71 14 0.874 5.76E-03 1.68E-03 SIAL AYEDEYSYFK 19 9 9 1 NA NA NA 19 9 9 1 NA NA NA SIAL TTSPPFGK 19 9 9 1 NA NA NA 0 0 0 0 ND ND ND SODM GDVTAQIALQPALK 154 69 71 14 0.156 4.45E-01 1.49E-01 151 67 70 14 0.168 4.24E-01 6.56E-02 SODM NVRPDYLK 154 69 71 14 0.156 4.45E-01 1.49E-01 3 2 1 0 0.615 4.19E-02 8.00E-03 SPON2 WSQTAFPK 63 30 31 2 NA NA NA 0 0 0 0 ND ND ND SPON2 YSITFTGK 63 30 31 2 NA NA NA 63 30 31 2 -

0.054 8.00E-01 1.19E-01

STAT1 TELISVSEVHPSR 38 18 17 3 NA NA NA 29 14 14 1 NA NA NA STAT1 YTYEHDPITK 38 18 17 3 NA NA NA 9 4 3 2 0.966 9.89E-04 4.69E-04 TBA1B AVFVDLEPTVIDEVR 119 60 56 3 NA NA NA 119 60 56 3 0.983 3.94E-04 2.97E-04 TBA1B EIIDLVLDR 119 60 56 3 NA NA NA 0 0 0 0 ND ND ND TBB3 ISVYYNEASSHK 158 72 71 15 0.123 5.05E-01 1.65E-01 158 72 71 15 0.991 1.10E-04 1.40E-04 TBB3 YLTVATVFR 158 72 71 15 0.123 5.05E-01 1.65E-01 0 0 0 0 0.349 1.71E-01 2.75E-02 TCPA IHPTSVISGYR 158 72 71 15 -

0.019 7.52E-01 2.35E-01 4 0 4 0 0.897 4.14E-03 1.32E-03

TCPA SSLGPVGLDK 158 72 71 15 - 7.52E-01 2.35E-01 154 72 67 15 0.705 2.66E-02 5.74E-03


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0.019 TCPQ DIDEVSSLLR 48 24 23 1 NA NA NA 0 0 0 0 ND ND ND TCPQ NVGLDIEAEVPAVK 48 24 23 1 NA NA NA 48 24 23 1 NA NA NA TCPZ GIDPFSLDALSK 6 1 5 0 NA NA NA 6 1 5 0 NA NA NA TCPZ GLVLDHGAR 6 1 5 0 NA NA NA 0 0 0 0 ND ND ND TENA GLEPGQEYNVLLTAEK 140 67 61 12 NA NA NA 140 67 61 12 0.469 8.55E-02 1.47E-02 TENA TVSGNTVEYALTDLEPAT

EYTLR 140 67 61 12 NA NA NA 0 0 0 0 ND ND ND

TENX DAQGQPQAVPVSGDLR 158 72 71 15 0.705 2.66E-02 1.38E-02 158 72 71 15 0.795 1.46E-02 3.47E-03 TENX YEVTVVSVR 158 72 71 15 0.705 2.66E-02 1.38E-02 0 0 0 0 0.878 5.46E-03 1.62E-03 TERA GILLYGPPGTGK 106 53 52 1 0.799 1.43E-02 1.00E-02 94 48 45 1 0.862 7.07E-03 1.96E-03 TERA LDQLIYIPLPDEK 106 53 52 1 0.799 1.43E-02 1.00E-02 12 5 7 0 0.851 8.10E-03 2.23E-03 TETN GGTLSTPQTGSENDALYE

YLR 158 72 71 15 0.367 1.54E-01 5.69E-02 118 53 50 15 0.977 6.23E-04 3.74E-04

TETN LDTLAQEVALLK 158 72 71 15 0.367 1.54E-01 5.69E-02 40 19 21 0 0.984 3.57E-04 2.92E-04 TFR1 LTVSNVLK 157 71 71 15 0.752 2.02E-02 1.29E-02 0 0 0 0 0.625 3.97E-02 7.73E-03 TFR1 SSGLPNIPVQTISR 157 71 71 15 0.752 2.02E-02 1.29E-02 157 71 71 15 0.962 1.14E-03 5.18E-04 TIMP1 GFQALGDAADIR 151 70 69 12 NA NA NA 151 70 69 12 0.789 1.55E-02 3.59E-03 TIMP1 SEEFLIAGK 151 70 69 12 NA NA NA 0 0 0 0 ND ND ND TNF12 AAPFLTYFGLFQVH 156 71 71 14 NA NA NA 156 71 71 14 0.644 3.62E-02 7.30E-03 TNF12 INSSSPLR 156 71 71 14 NA NA NA 0 0 0 0 ND ND ND TPIS VVFEQTK 157 72 71 14 0.967 9.52E-04 1.78E-03 157 72 71 14 0.927 2.46E-03 9.06E-04 TPIS VVLAYEPVWAIGTGK 157 72 71 14 0.967 9.52E-04 1.78E-03 0 0 0 0 0.988 2.01E-04 1.91E-04 TRFL FQLFGSPSGQK 48 17 22 9 NA NA NA 22 7 11 4 NA NA NA TRFL LRPVAAEVYGTER 48 17 22 9 NA NA NA 4 1 3 0 0.908 3.36E-03 1.12E-03 TRFL VPSHAVVAR 48 17 22 9 NA NA NA 5 0 0 5 NA NA NA TRFL YYGYTGAFR 48 17 22 9 NA NA NA 17 9 8 0 NA NA NA


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TSP1 GFLLLASLR 158 72 71 15 0.970 8.88E-04 1.78E-03 6 0 3 3 0.999 9.15E-06 2.99E-05 TSP1 GTSQNDPNWVVR 158 72 71 15 0.970 8.88E-04 1.78E-03 152 72 68 12 0.972 8.15E-04 4.13E-04 TTHY TSESGELHGLTTEEEFVEGI

YK 27 4 8 15 NA NA NA 27 4 8 15 0.996 1.83E-05 4.49E-05

TTHY VEIDTK 27 4 8 15 NA NA NA 0 0 0 0 ND ND ND TYPH ALQEALVLSDR 59 24 26 9 NA NA NA 0 0 0 0 ND ND ND TYPH TLVGVGASLGLR 59 24 26 9 NA NA NA 59 24 26 9 NA NA NA UGGG1 DLSQNFPTK 58 23 20 15 NA NA NA 58 23 20 15 NA NA NA UGGG1 FTILDSQGK 58 23 20 15 NA NA NA 0 0 0 0 ND ND ND UGPA LVEIAQVPK 97 49 45 3 NA NA NA 97 49 45 3 0.850 8.20E-03 2.23E-03 UGPA NENTFLDLTVQQIEHLNK 97 49 45 3 NA NA NA 0 0 0 0 ND ND ND VA0D1 LLFEGAGSNPGDK 13 1 0 12 NA NA NA 13 1 0 12 NA NA NA VA0D1 NVADYYPEYK 13 1 0 12 NA NA NA 0 0 0 0 ND ND ND VEGFC DLEEQLR 21 3 5 13 NA NA NA 21 3 5 13 NA NA NA VEGFC EAPAAAAAFESGLDLSDA

EPDAGEATAYASK 21 3 5 13 NA NA NA 0 0 0 0 ND ND ND

VEGFC FAAAHYNTEILK 21 3 5 13 NA NA NA 0 0 0 0 ND ND ND VEGFC NQPLNPGK 21 3 5 13 NA NA NA 0 0 0 0 ND ND ND VTNC AVRPGYPK 145 65 65 15 NA NA NA 0 0 0 0 ND ND ND VTNC DVWGIEGPIDAAFTR 145 65 65 15 NA NA NA 145 65 65 15 0.971 8.33E-04 4.15E-04 ZA2G EIPAWVPFDPAAQITK 158 72 71 15 NA NA NA 158 72 71 15 0.995 2.75E-05 6.22E-05 ZA2G WEAEPVYVQR 158 72 71 15 NA NA NA 0 0 0 0 ND ND ND

Note:

• Protein: UniProt protein name • Peptide: Peptide sequence


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• Protein detection: How many times the protein was detected in the 158 samples. It is also the sum of columns D, E and F. If any transition of the protein was detected in a sample, the protein was counted as detected in the sample. It is slightly higher than protein detection based on most intensive transition only.

• Protein detection in cancer: How many times the protein was detected in the 72 cancer samples. • Protein detection in benign: How many times the protein was detected in the 71 benign samples. • Protein detection in HPS: How many times the protein was detected in the 15 human plasma standard (HPS) samples. • Protein correlation: Pearson correlation between two peptides of the protein. Pearson coefficient was calculated between

tansitions having the highest median intensities of the corresponding peptides. If the two transitions were detected in less than five common samples, Pearson correlation was assigned to "NA".

• Protein p value: Empirical protein p value: See Supplementary Materials and Methods for more information. It was assigned to "NA" if the Pearson correlation was assigned to "NA".

• Protein q value: Protein q value: See Supplementary Materials and Methods for more information. It was assigned to "NA" if the Pearson correlation was assigned to "NA".

• Peptide detection: How many times the peptide was detected in the 158 samples. It is also the sum of columns K, L and M. If any transition of the peptide was detected in a sample, the peptide was counted as detected in the sample.

• Peptide detection in cancer: How many times the peptide was detected in the 72 cancer samples. • Peptide detection in benign: How many times the peptide was detected in the 71 benign samples. • Peptide detection in HPS: How many times the peptide was detected in the 15 HPS samples. • Peptide correlation: Pearson correlation between two transitions of the peptide. Pearson coefficient was calculated between

transitions having the highest median intensities of the peptide. If the two transitions were detected in less than five common samples, Pearson correlation was assigned to "NA". If the peptide was not detected, it was assigned to "ND".

• Peptide p value: Empirical peptide p value: See Supplementary Materials and Methods for more information. It was assigned to "NA" or "ND" if the Pearson correlation was assigned to "NA" or "ND".

• Peptide q value: Peptide q value: See Supplementary Materials and Methods for more information. It was assigned to "NA" or "ND" if the Pearson correlation was assigned to "NA" or "ND".

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Supplementary Materials for · Supplementary Materials: Materials and Methods Candidate biomarkers...

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