high sensitivity targeted quantitation of erk phosphorylation … · 2016. 1. 6. · high...

High Sensitivity Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry without Affinity Enrichment

Tujin Shi, Tao Liu, Matthew Gaffrey, Yuqian Gao, William Chrisler, Thomas Fillmore, Carrie Nicora, Marina Gritsenko, Chaochao Wu, Jintang He, Jia Guo, Rui Zhao, Ronald Moore, Richard D. Smith, David Camp, Karin Rodland, Steven Wiley, Wei-Jun Qian

Biological Sciences Division, Pacific Northwest National Laboratory

62nd ASMS Conference, Maryland, June 18, 2014

Outline

I. Background II. Proof-of-concept study

(direct quantification of individual site-specific ERK phosphorylation by PRISM-SRM)

III. Comparison of PRISM-SRM with IMAC-SRM IV. Quantification of ERK phosphorylation dynamics V. Conclusions

PRISM for much improved SRM sensitivity

Shi et al., Proc Natl Acad Sci USA 2012, 109, 15395-15400

10%

90%

(A) PRISM (high Pressure high Resolution Separation with Intelligent Selection and Multiplexing)

(B) LC-SRM

Significantly improved LOQs with PRISM

• Original development: Shi et al., Proc Natl Acad Sci USA 2012, 109,15395-15400 • PRISM-SRM without immunoaffinity depletion: Shi et al., J Proteome Res 2013, 12, 3353-3361 • Application for detection of AGR2 in urine and serum: Shi et al., J Proteome Res 2014,13, 875-882 • Application for detection of TMPRSS2:ERG fusion proteins in tissue: He et al., Mol Oncol 2014, in press

Targeted MS platform LOQs (starting material)

Human plasma/serum Mammalian cells

Conventional LC-SRM ~1 μg/mL (1 µL) ~5,000 copies/cell (?)*

PRISM-SRM ~1 ng/mL (2 µL) ~100 copies/cell (~25 µg)

IgY14-PRISM-SRM ~50 pg/mL (10 µL) N/A

*Beck et al., Mol Syst Biol 2011, 7: 549 *Kiel et al., J Proteome Res 2014, 13:300–313

PRISM publications:

PRISM-SRM is more sensitive than ELISA for EGFR measurement in MCF7 cells

ELISA

23.5 24.0 24.5 25.0Retention Time

0

1000

2000

3000

4000

5000

Inte

nsity

24.1

MDA-MB-231 Copies/cell: ~172,000 amol/µg: 1696

MCF7 Copies/cell: ~2,500* amol/µg: 21

Conventional LC-SRM

Hembrough et al., Clin Proteomics 2012, 9:5

23.5 24.0 24.5 25.0Retention Time

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Inten

sity (

10^6

)

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Heav

y

*Previously reported in similar range by: 1) Kosano et al., Cancer Res 1988, 48:6033–6; and 2) Roos et al., Proc Natl Acad Sci USA 1986, 83:991–5

23.5 24.0 24.5 25.0Retention Time

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nsity

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Endo

geno

us

ERK phosphorylation is ideal for evaluation of analytical platform performance

Moderate complexity: (two isoforms, ERK1 and ERK2; separate or concurrent phosphorylation on proximate T and Y residues in a TEY motif)

Well-studied both experimentally and theoretically

Challenging for measuring individual ERK phosphorylation isoforms with antibody-based assays

Targeted proteomics allows site-specific quantification of PTMs

However relatively large starting materials required for conventional SRM analysis (~1 mg cell lysate: Tong et al., Mol Cell

Proteomics 2009, 8, 2131-2144)

Proof-of-concept study

Surrogate peptides for ERK1/2 phosphorylation analysis

Proof-of-concept study

SRM Transitions

Q1 Q3

724.7 839.4 (y7+), 738.4 (y6+), 609.3 (y5+)

751.3 718.7 ([precursor – 98]3+), 821.4 ([y7 – 98]+), 927.9 ([y16 – 98]2+)

751.3 919.4 (y7+), 689.3 (y5+), 976.9 (y162+)

778.0 745.3 ([precursor – 98]3+), 901.4 ([y7 – 98]+), 967.9 ([y16 – 98]2+)

715.3 952.5 (y8+), 839.4 (y7+), 738.4 (y6+)

742.0 709.3 ([precursor – 98]3+), 934.5 ([y8 – 98]+), 738.4 (y6+)

742.0 919.4 (y7+), 818.3 (y6+), 689.3 (y5+)

768.7 736.0 ([precursor – 98]3+), 901.4 ([y7 – 98]+), 446.3 (y4+)

Sensitivity evaluation: the basal level and the stimulated level treated with 10 ng/mL EGF for 10 min Dose-response experiments: different EGF doses (0, 0.1, 0.3, 1.0, 3.0, 10 ng/mL) for either 10 min (peak activation) or 2 h (steady state) stimulations

TY-ERK1

pT-ERK1

pY-ERK1 pTpY-ERK1

TY-ERK2

pT-ERK2

pY-ERK2 pTpY-ERK2

25.5 26.0Retention Time

0200400600800

10001200

Inte

nsity

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050

100150200250300350

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0100200300400500600

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nsity

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nsity

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pTpY-ERK2 pY-ERK2 pT-ERK2

Molar abundance of individual ERK2 isoforms with standard deviation (n = 3)

PRISM-SRM enables direct quantification of site-specific ERK phosphorylation in HMEC cells

10 ng/mL EGF stimulation (10 min) Basal (no stimulation)

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Basal EGF (10 ng/mL)

Mol

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TY-ERK2pTpY-ERK2pY-ERK2pT-ERK2

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1015202530

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nsity

(10^

3)

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26.0Retention Time

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25.0 25.5 26.0Retention Time

02468

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nsity

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pTpY-ERK2 pY-ERK2 pT-ERK2

Ligh

t He

avy

Estimated LOQ for pT-ERK2: ~1000 copies per cell

PRISM-SRM vs. IMAC-SRM

~50 µg EGF-treated (10 ng/mL for 10 min) HMEC cell lysate digest

Addition of internal standard (heavy-isotope labeled peptides)

PRISM (~25 µg)

LC-SRM

IMAC (~25 µg)

LC-SRM

27.8 28.0 28.2 28.4Retention Time

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Inte

nsity

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3) 28.0

27.8 28.0 28.2 28.4Retention Time

0.00.51.01.52.02.53.0

(10^

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010203040506070

Inte

nsity

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3)

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0.00.51.01.52.02.53.03.5

(10^

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pTpY-ERK2 pT-ERK2

Hea

vyLi

ght


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nsity

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nsity

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010203040506070

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pTpY-ERK2 pT-ERK2

PRISM-SRM IMAC-SRM

PRISM-SRM provides ≥10-fold higher sensitivity than IMAC-SRM

Recovery of IMAC (relative to PRISM): pTpY (10.6%) pY (7.9%) pT (4.0%)

Ligh

t H

eavy

(small-sized samples)

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1000000

1500000

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2500000

3000000

PRISM IMAC

Peak

inte

nsity

(hea

vy IS

)

pTpY-ERK2pT-ERK2pY-ERK2

Distributive vs. processive phosphorylation model for ERK

Distributive model Processive model

Aoki et al., Proc Natl Acad Sci USA 2011, 108, 12675-12680

ERK phosphorylation dynamics as a result of EGF-induced dose response

Maximal ERK activation: 0.3-1 ng/mL EGF at peak activation (10-min); 3 ng/mL EGF at steady state (2-h) EGF dose at the peak activation close to physiological levels in humans (~0.5 ng/mL) Increase of pY-ERK2 mirrors that of pTpY-ERK2, but pT-ERK2 does not increase significantly

10-min stimulation 2-h stimulation

0%

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ativ

e ab

unda

nce

EGF (ng/mL)

TY-ERK2 pTpY-ERK2 pY-ERK2 pT-ERK2

80%

100%

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EGF (ng/mL)


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100%

0.0 0.2 0.4 0.6 0.8 1.0

Relative abundance changes (2-h vs. 10-min)

Time-dependent ERK2 phosphorylation (1 ng/mL EGF stimulation)

ERK phosphorylation dynamics as a result of EGF-induced dose response

Similar stoichiometry changes for pTpY-ERK2 and pY-ERK2 from peak activation to steady state; however pTpY-ERK2 changes more

0%

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100%

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nce

Time (min)


-40.0%

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EGF (ng/mL)


Time-course analysis: the activation pattern of pY-ERK2 is similar to that of pTpY-ERK2, but often with lower stoichiometry

ERK is phosphorylated in a processive, rather than distributive manner in mammalian cells

Processive (parallel)

Aoki et al., Proc Natl Acad Sci USA 2011, 108, 12675-12680

Computational simulation

Phos-tag WB data (HeLa cells)

Distributive (bell-shape)

PRISM-SRM data (HMEC cells)

Conclusions

PRISM-SRM enables sensitive, site-specific quantification of low level protein phosphorylation without affinity enrichment

Compared to IMAC, PRISM provides at least 10-fold improvement in SRM sensitivity

Phosphorylation dynamics data (dose responses and timecourse) confirms that ERK is phosphorylated in a processive manner in mammalian cells

PRISM-SRM has great potential for simultaneous quantification of multiple PTMs in a single analysis (i.e., without serial or parallel enrichment)

Acknowledgments

Contact information: [email protected]

National Institute of General Medical Sciences

Biomedical Technology Research Resource (BTRR)

mailto:[email protected]

http://www.nigms.nih.gov/

high sensitivity targeted quantitation of erk phosphorylation … · 2016. 1. 6. · high...

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