-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