Linear Peptide Response Non-Linear Peptide Response
Peptide Quality VennDiagram
100, 50, 25 fmol Isotope Dot Product > 0.95
Label-Free Peptide Dilution Experiment Distribution of Log-Ratios
Peptides were filtered if any IsotopeDotProduct > 0.5 (875 peptides remained, 138 filtered)
Rectangle Shaded Area is mean +/- one Standard Deviation
Fold Change
(relative to 100 fmol) Peptides were filtered if any IsotopeDotProduct > 0.5
(875 peptides remained, 138 filtered)
Dilution Ratios Peptides were filtered if any IsotopeDotProduct > 0.5 (875 peptides remained, 138 filtered)
Diagonal is histogram of log2peak Area
Off-diagonal is scatter plot between two log2 peak areas
ABRF-sPRG2013 Development and Characterization of a Proteomics Normalization Standard Consisting of 1,000 Stable Isotope Labeled Peptides
Christopher Colangelo1; Craig Dufresne2; Alexander Ivanov3; Antonius Koller4; Brett Phinney5; Kristie Rose6; Paul Rudnick7; Brian Searle8; Scott Shaffer9 1Yale University, New Haven , CT; 2Thermo Fisher Scientific, West Palm Beach, FL; 3Northeastern University, Boston, MA; 4Stony Brook University, Stony Brook, NY; 5University of California, Davis, CA; 6Vanderbilt University, Nashville, TN; 7National
Institute of Standards and Technology, Gaithersburg, MD; 8Proteome Software, Portland, OR; 9University of Massachusetts Medical School, Worcester, MA
A BR F
Participate in the sPRG2013 Study and have fun with quantitation and refining your analytical approaches!!! Visit www.abrf.org/sprg
Introduction Proteomics technologies are an integral part of biological and
clinical research. Significant contributions by the proteomics field
are driven by the diverse and advanced analytical approaches
employed to comprehensively characterize proteomes, including
quantitative analysis of proteome variations, modifications, and
interactions. The ABRF Proteomics Standards Research
Group (sPRG) functions to design and develop performance
standards and resources for mass spectrometry-based
proteomics applications. The sPRG is currently conducting
a study focused on generation of a standard for interassay,
interspecies, and interlaboratory normalization in label-free as
well as in quantitative stable-isotope label-based analyses. The
standard has been formulated as two mixtures: 1,000 stable
isotope 13C/15N-labeled synthetic tryptic peptides alone, and
peptides mixed with a tryptic digest from HEK 293 cell lysate.
Methods Sequences of synthetic peptides were derived from
approximately 552 proteins, conserved across proteomes of
commonly analyzed species: Homo sapiens, Mus musculus and
Rattus norvegicus. Peptides represent a wide range
of hydrophobicities and isoelectric points typical of complex
proteomics samples. The 1,000 isotope-labeled peptides were
spot-synthesized by jpt.com. Individual peptides were
reconstituted, combined and desalted by solid-phase extraction.
HEK cell lysates were prepared by RIPA lysis and sonication, and
proteins were methanol-precipitated, resolubilized, and digested
with Lys-C and trypsin. The HEK proteolytic peptide mixture was
desalted by solid-phase extraction. For the combined
formulation, 1 pmol of synthetic peptide was added to 5 μg of the
HEK peptide mixture. The synthetic peptide mixture and the
combined synthetic and HEK-derived peptide mixture
were characterized individually by sPRG members representing
multiple LC-MS/MS instrument platforms.
Conclusion The Proteomics Standards Research Group reports the progress
for the first year in development of an innovative proteomics
normalization standard, designed to represent proteins of various
concentrations and spanning three orders of magnitude. 1,000
isotope labeled peptides were synthesized and analyzed in the
laboratories of sPRG members with various instruments and
fragmentation methods. Greater than 99% of the peptides were
identified during the validation runs. In peptide dilution
experiments, the majority of peptides behaved as expected with a
linear response, however, a few peptides had a non-linear
response. The peptides were spiked into a HEK digest and
showed minimal variation in both retention time and peak area.
The sPRG is hopeful that the designed formulation will become a
valuable resource in various mass spectrometry-based proteomic
applications, including quantitative and differential protein profiling,
as well as general benchmarking (e.g. chromatographic retention
time) for LC-MS performance.
L S
L S M Y G V D L
T H S
K*
P F R*
K*= Lys (U-13C6;U-15N2), mass difference: + 8 Dalton
R*= Arg (U-13C6;U-15N4), mass difference: + 10 Dalton
ABRF sPRG Peptides
isotopically labeled, proteotypic peptides
that terminate with C-terminal heavy Arg/Lys
Instrument Platforms Used for Validation
Protein Identification of
ABRF Peptide Standards
Instrument LC IDA/SWATH Fragmentation
ABSciex 5600TT Waters NanoAcquity IDA QTOF
ABSciex 5600TT Waters NanoAcquity SWATH QTOF
Thermo Orbitrap-Elite Waters NanoAcquity IDA HCD
Thermo Orbitrap-Elite Waters NanoAcquity IDA CID(IT)
Thermo Orbitrap-XL Eksigent nano 2DLC IDA CID(IT)
Thermo Q-Exactive Waters NanoAcquity IDA HCD
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800x f
mo
l p
ep
tid
e a
rea
/10
0fm
ol
pe
pti
de
are
a
peptide
Individual Peptide Peak Area (relative to 100 fmol)
6fmol
12fmol
25fmol
50fmol
100fmol
Characterization of Peptide Standards
0
10
20
30
40
50
60
70
-2.7
-2.4
-2.1
-1.8
-1.5
-1.2
-0.9
-0.6
-0.3
0 0.3
0.6
0.9
1.2
1.5
1.8
2.1
# o
f p
ep
tid
es
Gravy scores
0
50
100
150
200
250
# o
f p
ep
tid
es
% hydrophobicity (Bull Breese Index)
0
50
100
150
200
6 8 10 12 14 16 18 20
# o
f p
ep
tid
es
peptide length
0
100
200
300
400
500
1 2 3
# o
f p
rote
ins
# of peptides
0
20
40
60
80
100
700
-750
850
-900
100
0-1
05
0
115
0-1
20
0
130
0-1
35
0
145
0-1
50
0
160
0-1
65
0
175
0-1
80
0
190
0-1
95
0
205
0-2
10
0
220
0-2
25
0
# o
f p
ep
tid
es
Molecular weight (Da)
- Linear Fit
- isotope dot product > 0.95
- isotope dot product > 0.8
- isotope dot product > 0.5
Acknowledgements The ABRF Proteomics Standards Research Group (sPRG) would
like to thank JPT Peptide Technologies GmbH for providing greatly
discounted synthetic peptides. We also thank Thermo Scientific
and Proteome Software for their generous technical support.
Data Processing and Analysis 50 Data Dependent (IDA) and 12 Data Independent (SWATH) LC-MS runs were performed using 4
types on instruments at 5 laboratories. MASCOT mgf files were generated from all 50 IDA files
using MSConvert (Proteomewizard) on ThermoScientific *.raw files and AB Sciex MS Data
Converter 1.3 *.wiff files. Protein Identification was performed using MASCOT 2.4 and a
concatenated ABRF peptide sequence and the human Swiss-Prot database. MASCOT dat files
were converted to XML and uploaded to Yale Protein Expression Database. Label-Free Precursor
Ion Analysis was performed using Skyline 1.4. Briefly, MASCOT dat files were imported to
generate a spectral library. Raw datafiles were integrated and the peak area results were exported
to csv file which were imported into Excel and R 2.14.2 for downstream biostatistical analysis.
ABRF Peptides
Both
HEK
Label-Free Peptides + HEK
Distribution of Peptide Ion Intensities Isotope dot product > 0.95
R² = 0.9838
0
25
50
75
100
125
150
0 25 50 75 100 125 150
AB
RF
Pep
tid
es +
HE
K
ABRF Peptides
ABRF Peptides
Both
HEK
Estimated Abundance (Middle) Protein: MATR3_HUMAN
Peptide: TEEGPTLSYGR (2+)
Estimated HEK amount – 125 fmol
Estimated Abundance (High) Protein: NPM_HUMAN
Peptide: VDNDENEHQLSLR (2+, 3+)
Estimated HEK amount – 1500 fmol
Protein ID of HEK All seventeen HEK LC-MS IDA runs
were combined and searched with
MASCOT against the human
Swissprot database. The search
results were filtered to 1.0% FDR on
the peptide spectrum match level,
resulting in 4,171 distinct proteins
from 126,173 distinct peptide
identifications greater than identity.
0
200
400
600
800
1000
1200
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Pep
tid
es >
Id
en
tity
(1%
FD
R)
% o
f A
BR
F P
ep
tid
es I
den
tifi
ed
Sample
% Coverage Peptide score > Identity score
Retention Times are Reproducible
Between Samples
ABRF Peptides
500fmol
HEK
1 ug
ABRF Peptides + HEK
500fmol + 1 ug