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