INTRODUCTIONPost translational modifications (PTMs) are crucial in controlling key aspects of protein function, including interactions in

signaling pathways. Identification and quantitation of the phosphorylation state of proteins involved in cell progression,

metabolism, growth, and disease is critical for the continued elucidation of cellular function1. Despite improvements in new MS

instrumentation, phosphoproteomic analyses still face challenges including low yield/specificity of phosphopeptide enrichment,

poor assignment of phosphorylation sites and low phosphorylation site stoichiometry. We have constructed a pool of about 146

heavy-labeled phosphopeptides from seven different signaling pathways that will enable the quantitation of 146

phosphopeptides in a single analysis using the optimized SMOAC phosphopeptide enrichment method couples to internal

standard triggered PRM (SureQuant) analysis.

Bhavin Patel1; Penny Jensen1; Aaron S. Gajadhar2; Sebastien Gallien3; Jae Choi1; Romain Huguet2; Graeme McAlister2; Derek Bailey2; Shannon Eliuk2; Markus Kellmann4; Tabiwang N. Arrey4; Alexander Harder4; Andreas Huhmer2; Kay Opperman1; John C Rogers1

1Thermo Fisher Scientific, Rockford, IL; 2Thermo Fisher Scientific, San Jose, CA; 3Thermo Fisher Scientific, Precision Medicine Science Center, Cambridge, MA; 4Thermo Fisher Scientific, Bremen, Germany

RESULTSABSTRACT Introduction

There is broad interest in quantifying dynamic protein phosphorylation states in cellular signaling pathways under different

conditions. We have combined SMOAC (Sequential enrichment of Metal Oxide Affinity Chromatography), 146 AQUA™

heavy-labeled phosphopeptide standards, and internal standard triggered targeted MS to evaluate changes in

phosphorylated protein abundance under different stimulation conditions. The specific phosphopeptides have been

chosen to cover biologically interesting phosphosites from several different signaling pathways.

Methods

We developed an assay containing a pool of 146 AQUA heavy-labeled phosphopeptides from 89 signaling proteins.

HeLa/A549 cells were grown with different stimulation conditions (hIGF-1/hEFG) before in-solution digestion. One

milligram of each cell digest spiked with phosphopeptides standard was subjected to Thermo Scientific™ Hi-Select TiO2

phosphopeptide enrichment kit (PN#A32993). TiO2 flow-through/wash fractions were enriched with the Hi-Select Fe-NTA

phophopeptide enrichment kit (PN#A32992). Both eluents were combined before LC-MS analysis using Thermo

Scientific™ Dionex™ nanoLC™ system coupled to modified Orbitrap mass spectrometers. To ensure optimal

measurement of each target, a novel targeting Thermo Scientific™ SureQuant™ method was performed where real-time

heavy peptide detection triggered high-sensitivity measurement of endogenous targets. Data analysis was performed with

Proteome Discoverer and Skyline software.

Preliminary Data

We have previously described our optimized SMOAC phosphopeptide enrichment method and we have shown with that

method significant improvement in the number of phosphopeptides identified. In this study, we developed a targeted assay

based upon 146 AQUA heavy-isotope phosphopeptide standards (96 serine, 26 threonine and 36 tyrosine modified

peptides). More than 80% of peptides were quantified with SureQuant method. The phosphopeptide standards spiked into

stimulated HeLa and A549 cell digest, followed by enrichment using the SMOAC method, allowed quantitation of

endogenous phosphopeptides by a directed discovery (DDA with inclusion list and DIA) method. With an adapted internal

standard triggered PRM method (SureQuant), using the modified Orbitrap MS instruments, we quantified multiple

phosphopeptides in the SMOAC enriched HeLa and A549 stimulated digest. This SIL-triggered targeted analysis allowed

much better quantitation of signaling pathway phosphorylated proteins by enhancing the detectability of targets and

significantly improving measurement reproducibility across the different stimulation conditions. This targeted

phosphopeptide assay coupled with SMOAC method and novel MS acquisition approach provided excellent quantitation,

specificity and selectivity for signaling pathway analysis.

Novel Aspect

This phosphopeptide standard with novel targeted MS analysis allowed quantitation of phosphorylation changes from 89

signaling pathway proteins.

CONCLUSIONS▪ Multi-pathway phosphopeptide profiling with SureQuant IS triggered method utilizes the presence of

synthetic isotopically labeled heavy peptides to enable sensitive and reproducible target multiplexing

measurement of about 134 phosphopetides in a single analysis with accurate and precise quantitation.

▪ The combination of EasyPep MS sample prep kit and SMAOC phosphopeptide enrichment method

followed by IS guided SureQuant method presents a new paradigm for signaling pathway analysis

involving PTMs.

▪ Multi-pathway phosphopeptide quantitation using the spiked-in internal standard and targeted MS

method provides easy interpretation of complex phosphopeptide signatures and positional isomers.

REFERENCE1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the use of targeted inhibitors in

cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

ACKNOWLEGEMENTWe would like to acknowledge ABRF sPRG group members and 2018-2019 study participants/sponsors

for providing DIA data.

For Research Use Only. Not for use in diagnostic procedures.

TRADEMARKS AND LEGAL INFORMATION© 2019 Thermo Fisher Scientific Inc. All rights reserved. AQUA is a trademark of Harvard Medical School.

SEQUEST is a trademark of the University of Washington. All other 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.

Quantitative, comprehensive multi-pathway signaling analysis using an optimized phosphopeptide enrichment method

combined with an internal standard triggered targeted MS assay

MATERIALS AND METHODS

Figure 1. Generating a 146 Synthetic Phosphopeptide Standard for Multipathway Analysis

Cell Culture

HeLas3 (PN#CCL-2.2) and A549 (PN#CCL-185) cells were purchased from ATCC and cultured in Life Technologies™

Gibco™ SMEM or FK-12 with 10% Fetal Bovine Serum complete media. hIGF-1 was acquired from Thermo Fisher

Scientific and hEGF was acquired from Cell Signaling Technology. HeLaS3 and A549 cells at approximately 80%

confluency were treated for 15 minutes with hEGF and hIGF-1 respectively, after 24 hour serum starvation using

appropriate media plus 0.1% charcoal-stripped FBS. Cells were lysed with TEAB/SDS plus universal nuclease as lysis

buffer. Thermo Scientific™ Pierce™ BCA Protein Assay (PN#23225) was performed for protein quantitation.

MS Sample Preparation and Phosphopeptide Enrichment

Thermo ScientificTM EasyPep™ Mini MS Sample Prep kit reagents (PN#A40006) with modified scale up protocol was

used to prepare digest from HeLa (+hEGF) and A549 (+hIGF-1) cell lysate. The optimized SMOAC method was used for

phosphopeptide enrichment. Briefly, 100fmol of 146 phosphopeptides standard was spiked-in to one milligram per

replicate of stimulated A549 or HeLa digest. Spiked-in digest was subjected to HiSelect TiO2 phosphopeptide enrichment

kit (PN#A32993) and the TiO2 eluent was saved for MS analysis. The TiO2 flow-through and wash fractions were pooled,

and the phosphopeptides were enriched by HiSelect Fe-NTA phosphopeptide enrichment kit (PN#A32992). Replicate

samples for all TiO2 enrichment steps and Fe-NTA enrichment steps were combined into separate pooled samples. After

SMOAC, phosphopeptides were cleaned off-line using the Pierce C18 Spin Tips (PN#84850) followed by peptide

quantitation using the Thermo Scientific Pierce Colorimetric Peptide Assay (PN#23275).

LC-MS Analysis

For the LC-MS analysis using DDA or DIA method, Thermo Scientific™ EASY-Spray™ C18 LC column (2 µm particle

size) to separate peptides with a 5-30% acetonitrile gradient over 120 minutes at a flow rate of 300 nL/min. Spectra were

acquired on an Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System or EASY-nLC™ 1200 system coupled

to Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.

LC-MS analysis of PRM or SureQuant method was performed with an EASY-nLC 1200 coupled to Thermo Scientific™

Orbitrap Exploris™ 480 and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ Mass Spectrometers. The overall SureQuant

workflow consists of two steps: (i) A ‘Survey run’ experiment to determine optimal precursor charge states, establish

corresponding fragment ions, and determine the apex intensity of the IS, (ii) SureQuant experiments where the instrument

monitors for the optimal m/z and triggering intensity (1% of apex) of the IS trigger peptides and upon its detection,

dynamically performs a high-resolution high-sensitivity MS2 analysis of the corresponding endogenous target. For both

Survey and SureQuant analysis, 60 min gradients, at 400nL/min were performed. 600fmol of the 146 phosphopeptide IS

mixture enriched from stimulated A549 or HeLa digest was used for the survey run analysis to determine intensity

thresholds for subsequent SureQuant analysis.

Data Analysis

For DDA data analysis, Thermo Scientific™ Proteome Discoverer™ 2.2 software was used to search MS/MS spectra with

the SEQUEST™ HT search engine with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da.

Static modification included carbamidomethylation (C). Dynamic modifications included methionine oxidation and

phosphorylation (S,T,Y). PhosphoRS node was used for site localization. For targeted PRM or SureQuant data analysis,

Skyline software (University of Washington) was used to develop targeted assay and measure light/heavy ratio and

calculate concentrations from unknown samples.

9%

12%

23%

18%

10%

16%

11% AMPK signaling

Death and apoptosis signaling

EGFR/HER signaling

Insulin/IGF-1 signaling

mTOR signaling

PI3K/AKT signaling

Stress (p38/SAPK/JNK) signaling

Figure 2. SMOAC Enriched Multi-Pathway Phosphopeptide SureQuant Profiling

Balanced across

chromatographic retention time

for use as internal RT standard

Phospho-peptides span wide dynamic

range in previous DDA experiments

(Phosphopedia database)

Interpretation of complex phosphopeptide signatures and positional isomers is aided by heavy standards

and DIA/targeted MS acquisition methods.

Phospho enrichment = CST IMAC

LC-MS/MS acquisition = 8X gas

phase fractionated DIA injections each

with 4 m/z fully overlapping windows

spanning 100 m/z across a 400-1200

total m/z range

A549 (+hIGF) HeLa (+hEGF) A549 (+hIGF) HeLa (+hEGF)

Figure 4. Data Dependent Analysis (DDA) of SMOAC Enriched Multi-Pathway Phosphopeptides

SureQuant method

IS peptides (trigger)ENDO

PeptidesPseudo Spectral

Matching✓

‘QUANT MODE’

High Quality MS2

of Target

✓ ✓ ✓ ✓ ✓

‘WATCH MODE’

Fast, Low Res MS2

of Trigger

1

4

5

2

3

Figure 7. SureQuant Acquisition Method Delivers Intelligent Detection of Targets

RT: 0.00 - 140.00 SM: 7B

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relat

ive A

bund

ance

19.81434.88

129.08702.37

66.67670.31

77.49625.34 82.64

734.70116.72780.40

47.17520.73

98.58755.01

114.92622.97

105.44793.74

92.83594.81

44.99698.99

60.30757.34

84.80755.01

54.32525.73

33.79403.52

75.82550.24

103.71755.34

40.33650.28

24.09560.27

91.39943.76

48.77657.29

74.89787.84 113.11

1048.48 133.39376.26

32.04542.23

120.63993.219.81

424.7226.50595.30

7.15487.22 17.88

317.70

14.54318.18

2.46325.23

139.75311.14

NL: 7.14E8

Base Peak F: ms MS Easy1200_DDA_QEHF_150phosAQUAbs_250fmolOC_Gr2_11Mar2019_R2

Figure 3. LC-MS Analysis of 146 Synthetic Phosphopeptide Standard

SMOAC enriched phosphopeptides from A549 (+hIGF) and HeLa (+hEGF) and nanoLC-MS analysis

using the DDA with inclusion list method resulted in identification of 114 heavy IS phosphopeptides.

Examples of phosphopeptides without interference (A) and with interference (B) from enriched samples.

Figure 5. Data Independent Analysis (DIA) of IMAC Enriched Multi-Pathway Phosphopeptides

Tryptic peptides

Multi-Pathway

Phosphopeptides

Standard

Mix

HiSelectTM

TiO2

HiSelectTM

Fe-NTA

Sample

Enriched heavy and endogenous

phosphopeptides

✓✓ ✓ ✓ ✓✓

Sequential Metal Oxide Affinity

Chromatography (SMOAC)

LC-MS Analysis

A. B.

SureQuant method

Figure 6. PRM Analysis of SMOAC Enriched Multi-Pathway Phosphopeptides

DIA analysis of IMAC enriched phosphopeptides from HeLa (+hEGF) showed better quantitation

compared to precursor-level (DDA) analysis. About 50 light and 124 heavy IS phosphopeptides were

quantitated across 4 different core labs using standardized DIA workflow (sPRG 2018-2019 study).

Orbitrap Exploris™ 480 Q Exactive™ HF

Heavy

Light

LYN (Y397): VIEDNEYTAR

PRM analysis of SMOAC enriched phosphopeptides from A549 (+hIGF) and HeLa (+hEGF) resulted in

accurate and precise quantitation of 134 heavy IS phosphopeptides. Improved signal to noise and

sensitivity was observed with new Orbitrap ExplorisTM 480 (Figure 6). Differential expression of many

phosphopeptides observed between two cancer cell lines (data not shown).

MAP2K4:S80

TSC2:S939

PLCG1:S1248

CHEK1:S317

TBC1D4:S318

TP53:S315

LYN:Y397

IRS1:S1101

RPS6KB1:T444_2

MEF2A:S408

AKT1S1:T246

LMNA:S22

LCK:Y394

SIRT1:S27

PTK

2:Y576

ACLY:S455

PLCB3:S537

PXN:Y118

FOXO3:S294

STAT3:Y705

GSK3A:S21

CHEK1:S280

IRS1:S341_2

RIPK2:S176

MDM2:S166

HSPB1:S82

ACACA:S80

IGF1R/IR:Y1135

PAK1:S144

TBC1D4:S588

CAV1:Y14

PTK2:Y925

MAPK3:Y204

RAF1:S259

IRS1:S323_2

PPP1CA:T320

ERBB3:Y1328

CAMKK2:S511

BRAF:S446

JUN:S63

A549 + IGF- 4. 24331826 - 2. 198924141 - 3. 750493979 - 6. 546245393 - 8. 429731384 - 6. 254289378 - 4. 943416472 - 2. 289827252 - 8. 333516069 - 2. 276485124 - 0. 564691448 2. 381864764 - 1. 264997807 - 6. 687799537 0. 543099892 3. 857752136 - 0. 943139057 - 3. 075824085 - 4. 594225422 - 3. 415807142 0. 527270557 - 3. 799872346 - 8. 28771238 - 3. 752437003 - 1. 930160375 6. 601176506 - 1. 20889443 - 5. 580353247 - 1. 145924538 - 7. 702749879 - 5. 622376462 - 8. 117787378 4. 498461511 - 19. 93156857 - 19. 93156857 - 3. 089267338 - 7. 454822365 - 6. 895394957 5. 327205907 - 2. 891107598

HeLa + EGF- 5. 28208783 - 2. 391379976 - 2. 019755295 - 6. 947862377 - 5. 779917739 - 10. 96578428 - 5. 66566056 - 9. 28771238 - 8. 532824877 - 1. 75389599 - 1. 316887478 1. 520145627 - 3. 075824085 - 6. 717856771 - 6. 532824877 1. 524414776 - 5. 546245393 - 11. 28771238 - 3. 221623189 4. 080751453 - 0. 777195439 - 2 - 19. 93156857 - 4. 993091631 - 3. 45324133 4. 696322167 - 1. 748068873 - 11. 28771238 - 5. 442222329 - 5. 673002535 - 11. 70274988 - 19. 93156857 - 0. 795358434 0. 304978236 - 7. 078259014 - 19. 93156857 - 11. 28771238 - 5. 836501268 - 0. 220278019 - 5. 423526235

Log2 ratio to heavy

PTK2 Y576TSC S939

HeLa + EGF A549 + IGF HeLa + EGF A549 + IGF

Figure 8. SureQuant Analysis of SMOAC Enriched Multi-Pathway Phosphopeptides

Figure 9. Benefits of Heavy Multi-Pathway Phosphopeptides Standard

SureQuant analysis of SMAOC enriched phosphopeptides provided accurate and precise quantitation

in a single MS run. About 60 light and 134 heavy IS phosphopeptides were quantitated using

SureQuant method on Orbitrap ExplorisTM 480. PO65550-EN0519S