-80 cv
-60 cv
-40 cv
Figure 4. Showing the importance of selecting
the right compensation voltage for three different
peptides. (*) representing the optimal CV.
Figure 5. Spectra of peptide
DQITAGNAAR without FAIMS
and with FAIMS, and the
peptides respective –y ion
fragmentation series.
DAENHEAQLK
-80 cv
-60 cv
-40 cv
NQGGGLSSSGAGEGQGPK
VPVHDVTDASK
**
DQITAGNAAR y8
y7
y6
y5
y7
No FAIMS FAIMS
-80 cv
-60 cv
-40 cv
*
Kerry Hassell1, Mary Blackburn2, Michael Belford2, Michael Volny2, Scott Peterman2, Romain Huguet2; 1 265 Davidson Ave, Suite 101, Somerset, NJ 07783, 2 355 River Oaks Parkway, San Jose, CA 95112
Developing the Research to Routine Workflows with FAIMS: Automating Large-scale SRM Method Creation for
Routine HeLa Peptide Screening
RESULTS
Discovery HRAM Experiments
The discovery method was used to fully characterize the HeLa digest. Replicate sample injections
using single CV settings significantly increases the protein coverage from 310 proteins without FAIMS
to over 500 proteins with FAIMS. Example of the base peak is shown in Figure 3, demonstrating the
improvement using FAIMS.
ABSTRACT
Purpose: To demonstrate a discovery to quantitation work flow for peptide panel using FAIMS.
Methods: Used high resolution data to pick peptides for proteins and converted them to an SRM
table to be analyzed on a triple quadrupole mass spectrometer.
Results: Improvement of 95% of the transitions monitored was seen using the new FAIMS source
which lead to lower limits of detection.
INTRODUCTION
Highly multiplexed protein panels are developed to enable routine sample screening while
maintaining high throughput. The challenge to creating an analytically robust SRM method is
determining which peptides to select per protein and creating the resulting SRM table for confident
data acquisition. Each protein added to the target list increases total SRM count by 9 quickly causing
acquisition challenges on triple quadrupole mass spectrometers as most proteotypic peptides cluster
into small hydrophobicity groups. To increase the selectivity space, we have incorporated a novel
source, field asymmetric waveform ion mobility spectrometry (FAIMS) interface for both profiling and
screening to increase the selectivity metrics for an SRM method monitoring over 300 HeLa proteins
in 60 minutes.
MATERIALS AND METHODS
Sample Preparation
A stock solution of Thermo Scientific™ Pierce™ HeLA Protein Digest was used for all experiments,
injection 200 ng of HeLa. Pierce Retention Time Calibration (PRTC) mixture was spiked in at 5
fmol/uL.
LC/MS
HeLa proteome profiling was performed using an Thermo Scientific™ Easy-nLC 1200™, Thermo
Scientific™ Orbitrap Tribrid™ mass spectrometer with a Thermo Scientific™ FAIMS Pro™ interface.
A HeLa digest was injected and analyzed using a single compensation voltage (CV) setting by
standard DDA methods and repeated for eight different CV settings. Each RAW file was processed to
create a data matrix of proteins and peptides, retention time, CV, and precursor and product ion
distribution profiles.
Data Analysis
A routine was created to construct a scheduled SRM table for the top 300 HeLa proteins using over
2500 SRM transitions. The SRM table was imported into a triple quadrupole mass spectrometer,
Thermo Scientific™ TSQ Altis™ with the FAIMS Pro interface and evaluated for analytical
performance.CONCLUSIONS▪ Field asymmetric waveform ion mobility spectrometry (FAIMS) can be used in a discovery
environment to determine a peptide screening panel. These peptides can be put into a targeted panel
to be monitored by a triple quadrupole mass spectrometer. The use of the FAIMS Pro interface
increases signal-to-noise of these peptides.
▪ Different compensation voltages can be run early in the workflow process to determine the optimal
voltage this is than translated to the targeted panel without additional optimization.
▪ Due to the improvement of signal-to-noise, linearity of calibration curves is improved and lower
LLOQs can be obtained.
▪ Further analysis will be done to inquire on looking at the number of transitions per peptide and
determining what protein expression levels can be monitored
For Research Use Only. Not for use in diagnostic procedures.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo
Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these
products in any manner that might infringe the intellectual property rights of others.
Peptide/Protein Selection – Pinnacle Software
Pinnacle software (Optys Tech Corporation) offers capabilities of searching routing (either spectral or
sequence matching), new library creation, facilitating user-defined protein selections, establishment
of peptide selection rules, and building the PRM/SRM assay. Example of the peptide selection is
shown in Figure 6.
Pinnacle
Pooled, fractionated, and/or individual samples
Protein IDCV Fractionation
Orbitrap Fusion Lumos MS with FAIMS Pro
Consolidated Spectral Library
Creation
Data Dependent Analysis
(DDA)
SRM selection
and refinement
➢ Retention Time
Scheduling
➢ Final Method
Figure 1. From left to right, the new FAIMS Pro interface, Easy Nano 1200, and TSQ Altis triple
quadrupole mass spectrometer.
0
100000
200000
300000
400000
NOFAIMS
FAIMS40
FAIMS45
FAIMS50
FAIMS55
FAIMS60
FAIMS65
FAIMS70
FAIMS75
FAIMS80
NQGGGLSSSGAGEGQGPK
0
300
600
Not Improved Improved
FAIMS - Peptide Improvement
0
2000
4000
6000
8000
10000
NOFAIMS
FAIMS40
FAIMS45
FAIMS50
FAIMS55
FAIMS60
FAIMS65
FAIMS70
FAIMS75
FAIMS80
DSDLSHVQNK
No FAIMS FAIMS
no FAIMS with FAIMS
Loss of linearity due
to increased noise in
interfering peaks
With FAIMS, linearity is
maintained for low
concentration calibrators
Figure 2. Workflow of discovery to routine quantitation of a large peptide panel.
Figure 3. HRAM spectra of the overall base peak chromatogram with and without FAIMS. The inset
demonstrates a low level peptide gaining intensity with FAIMS
Figure 7. SRM table
of peptides targeted
in HeLa analysis.
Optimized CVs are
represented in the far
right column.
Figure 8. Visualization tool with the method editor showing the number of transitions per
time as well as the dwell times of different precursor masses over the chromatographic
timescale.
Figure 6. Screenshot of workflow of the selection of proteins/peptides from Pinnacle.
Figure 13. Consistency of source and instrumentation. The figure to the left is HRAM data at
different compensation voltage, whereas the right figure is compensation voltages on a triple
quadrupole. The same CV is ideal for this peptide on both instruments
Figure 8. PRTC total ion current of peak intensity with and without FAIMS.
Figure 9. Peptide LTILEELR (heavy labeled) spiked into HeLa in
concentration range 1 attomol/uL – 100 fmol/uL linearity remains
consistent with FAIMS across linear range.
Figure 7. Comparison of peak area of no FAIMS, red, to different compensation voltages
highlighted in blue.
Figure 11. Number of transitions that were
improved versus not improved
no FAIMS with FAIMS
Slope = 1.94e-4
R2 = 0.9975Slope = 2.02e-4
R2 = 0.9981
Figure 10. Peptide LTILEELR (heavy labeled) spiked into HeLa in concentration range 1
attomol/uL – 1 fmol/uL at low concentration interferences are present and with FAIMS the
linearity is improved at lower concentrations.
Rela
tive
Ab
un
da
nce
-80 cv
-60 cv
-40 cv
Optimization of compensation voltages is demonstrated in Figure 4 for three different peptides,
showing the importance of using the correct CV for individual peptides.
Figure 5 is an example of the improvement of peptide fragmentation when using FAIMS.
In addition, the resulting data are used to create a four-dimensional library that consists of the protein
and corresponding peptides, and for each peptide, the measured retention time, CV setting, and
optimal precursor m/z value and product ion distribution.
Targeted Experiments – Triple Quadrupole Mass Spectrometry
The addition of FAIMS enhances the selectivity and sensitivity of peptides, increasing the number of
available peptides per targeted protein and resulting in more options to be considered in creating the
scheduled SRM table.
The set of experiments was performed on a high-end triple quadrupole mass spectrometer capable
of acquiring robust data with less than 5 msec dwell times per SRM transition and variable dwell
time settings per SRM transition. The SRM table is presented in Figure 7. For each peptide one to
three transitions were selected to monitor; this resulted in many transitions to be monitored. In order
to determine if there was enough dwell time, a visualization tool in the method editor software was
used to schedule windows, and is shown in Figure 8.
For the different protein groups targeted, the optimal peptides were selected based on relative
response in the discovery method, but more importantly on the retention time and CV setting as the two
values were used to create the final SRM table. Peptides were grouped into overlapping retention time
and CV bins to maximize duty cycle while maintaining analytical performance. FIG. 7 depicts the
variation of transmitted ion abundance with CV value for two peptides.
PRTC, a well known heavy labeled peptide standard was used to show overall intensity improvement
using FAIMS and the results can bee seen in Figure 8. The standard was than made into a dilution
series to study if linearity or lower limit of detection can be obtained using FAIMS. Figure 8 illustrates
that the linearity is not affected by FAIMS, and Figure 9 demonstrates the ability to reach a lower LLOQ
because of diminishing interferences in the quadrupole’s isolation window.
Of the 150 proteins monitored there was 95% improvement of peptide signal, with varying results of
improvement.
95% improvement
0.00E+00
2.50E+07
5.00E+07
7.50E+07
AEE
LAA
R
IQST
PV
K
LSEA
MA
R
LQTV
AEK
GA
AP
TPP
GK
YCD
PD
SYH
R
AN
EDEL
K
SDEM
NV
K
NR
PP
LPA
GTN
SKLS
VG
TNEK
LTEG
MSG
R
TSTA
PLD
R
MEN
AH
TK
ETEA
ALS
R
DA
TYTS
AR
RP
LED
GD
QP
DA
K
LGQ
EATV
GK
SHEA
EVLK
TTG
EGTS
LR
THLS
LSH
NP
EQK
HSQ
YHV
DG
SLEK
LYEE
GSN
K
IAEM
ETQ
K
TDTG
EPM
GR
YAD
LSH
NR
LNQ
MD
QD
K
AEV
SIQ
NN
K
TIQ
EMQ
QK
RP
FDP
ND
R
TTTE
SEV
MK
EAP
AQ
PA
PEK
EREM
AEM
R
AV
TEQ
GH
ELSN
EER
SCSP
ELQ
QK
KLN
VTE
QEK
AA
ATA
EEP
DP
K
AIQ
GG
TSH
HLG
QN
FSK
EAEN
PEG
EEK
TDG
CH
AYL
SK
HES
QM
DSV
VK
HQ
GV
MV
GM
GQ
K
LNEC
VD
HTP
K
AQ
QEL
EEQ
TR
HLT
HA
QST
LDA
KH
VP
GG
GN
VQ
IQN
K
AQ
AV
SED
AG
GN
EGR
NED
EEEE
EEEK
TCV
AD
ESH
AG
CEK
HEC
QA
NG
PED
LNR
NQ
GG
YGG
SSSS
SSYG
SGR
ITEA
PA
SEK
QV
HP
DTG
ISSK
Normalized Area
Figure 12. Different peptides showing the wide range of area response.
0.00E+00
2.50E+05
5.00E+05
AVPTDEAR FVSEDDR DPTNLQGK MAAHTMGNATVGR
PO65555-EN0519S
No FAIMS
FAIMS
RT :3.32-140.01
0
20
40
60
80
100
Re
lative
Abundance
0
20
40
60
80
100
Rela
tive
Abundance
9.31
24.73
83.9720.32 30.5479.86
34.10 125.0986.3746.82 67.7519.88103.55 111.25 127.7593.45
9.88
24.7015.39
81.1347.06 68.1928.0420.65 85.46
124.9177.3757.99113.19105.38
138.4995.64
20 40 60 80 100 120 140
Time (min)
NL: 5.62E8
Base Peak m/z=
200.0000-
1500.0000 MS
SMP_HeLa_200ng_
40_60_80_constant_
20190203215452
NL: 7.14E8
Base Peak m/z=
200.0000-
1500.0000 MS
HeLa_0_7Da_Iso_03
RT :0.00-47.05
0
20
40
60
80
100
Re
l ati
ve
Ab
un
da
nce
0
20
40
60
80
100
Re
l ativ
eA
bu
nd
an
ce
RT: 22.30
AA: 1.865E7
RT: 23.09
AA: 4.205E5RT: 44.38
AA: 6.453E4
RT: 22.45
AA: 1.560E7
RT: 27.68
AA: 2.960E5
RT: 23.16
AA: 3.314E5
0 5 10 15 20 25 30 35 40 45
Time (min)
NL: 1.86E6
m/z= 747.8043-747.8117 MS
F: FTMS + p NSI cv=-60.00
Full ms [375.0000-1500.0000]
SMP_HeLa_200ng_40_60_80_c
onstant_20190203215452
NL: 1.79E6
m/z= 747.8043-747.8117 MS
F: FTMS + p NSI Full ms
[375.0000-1500.0000]
HeLa_0_7Da_Iso_03
RT :0.00-140.01
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
9.46
10.30 10.60
9.44
9.44
7.5 8.0 8.5 9.0 9.5 10.0 10.5
Time (min)
NL: 3.14E6
m/z= 571.7726-571.7784 MS
F: FTMS + p NSI cv=-60.00
Full ms [375.0000-1500.0000]
SMP_HeLa_200ng_40_60_80_c
onstant_20190203215452
NL: 2.64E3
m/z= 571.7726-571.7784 MS
F: FTMS + p NSI cv=-40.00
Full ms [375.0000-1500.0000]
SMP_HeLa_200ng_40_60_80_c
onstant_20190203215452
NL: 2.64E3
m/z= 571.7726-571.7784 MS
F: FTMS + p NSI cv=-40.00
Full ms [375.0000-1500.0000]
SMP_HeLa_200ng_40_60_80_c
onstant_20190203215452
RT :0.00-33.17
0
20
40
60
80
100
Rela
tive
Abun
dance
0
20
40
60
80
100
Rela
tive
Abundance
0
20
40
60
80
100
Re
lative
Abund
ance
12.34
12.6112.12
12.02
12.6611.65
0 5 10 15 20 25 30
Time (min)
NL: 1.13E4
TIC MS F:
DSDLSHVQNK(+2):+ c NSI
cv=-80.00 SRM ms2
571.775 [655.267-655.269,
754.336-754.338]
HeLa_200ng_FAIMS_80
NL: 1.48E4
TIC MS F:
DSDLSHVQNK(+2):+ c NSI
cv=-60.00 SRM ms2
571.775 [655.267-655.269,
754.336-754.338]
HeLa_200ng_FAIMS_60
NL: 0
TIC MS F:
DSDLSHVQNK(+2):+ c NSI
cv=-40.00 SRM ms2
571.775 [655.267-655.269,
754.336-754.338]
HeLa_200ng_FAIMS_40_I