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
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Inten
sity (
10^6
)
24.1
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
0
100
200
300
400
500
Inte
nsity
(10^
3)
24.3
23.5 24.0 24.5 25.0Retention Time
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Inte
nsity
(10^
6)
24.3
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
25.8
25.5 26.0Retention Time
050
100150200250300350
(10^
3)
25.7
25.0 25.5Retention Time
0100200300400500600
Inte
nsity
25.2
25.0 25.5Retention Time
0
50
100
150
200
250
(10^
3)
25.2
29.0Retention Time
0
50
100
150
200
250
Inte
nsity
29.0
29.0Retention Time
0
50
100
150
200
(10^
3)
29.0
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)
0
50000
100000
150000
200000
250000
Basal EGF (10 ng/mL)
Mol
ecul
es/c
ell
TY-ERK2pTpY-ERK2pY-ERK2pT-ERK2
26.0Retention Time
05
1015202530
Inte
nsity
(10^
3)
26.1
26.0Retention Time
0
100
200
300
400
500
(10^
3)
26.1
25.0 25.5 26.0Retention Time
02468
1012
Inte
nsity
(10^
3)
25.6
25.0 25.5 26.0Retention Time
050
100150200250300350
(10^
3)
25.6
30.5Retention Time
0
100
200
300
400
500
Inte
nsity
30.4
30.5Retention Time
0
50
100
150
200
250
(10^
3)
30.4
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
0
5
10
15
20
Inte
nsity
(10^
3) 28.0
27.8 28.0 28.2 28.4Retention Time
0.00.51.01.52.02.53.0
(10^
6)
28.0
23.8 24.0 24.2Retention Time
010203040506070
Inte
nsity
(10^
3)
24.0
23.8 24.0 24.2Retention Time
0.00.51.01.52.02.53.03.5
(10^
6)
24.0
pTpY-ERK2 pT-ERK2
Hea
vyLi
ght
24.5 25.0Retention Time
0
2000
4000
6000
8000
Inte
nsity
24.8
24.5 25.0Retention Time
050
100150200250300
(10^
3)
24.8
28.4 28.6Retention Time
0
50
100
150
200
250
Inte
nsity
28.5
28.4 28.6Retention Time
010203040506070
(10^
3)
28.5
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)
0
500000
1000000
1500000
2000000
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%
20%
40%
60%
80%
100%
0.0 2.0 4.0 6.0 8.0 10.0
Rel
ativ
e ab
unda
nce
EGF (ng/mL)
TY-ERK2 pTpY-ERK2 pY-ERK2 pT-ERK2
80%
100%
0%
5%
10%
0.0 0.2 0.4 0.6 0.8 1.0
0%
20%
40%
60%
80%
100%
0.0 2.0 4.0 6.0 8.0 10.0
Rel
ativ
e ab
unda
nce
EGF (ng/mL)
TY-ERK2 pTpY-ERK2 pY-ERK2 pT-ERK2
0%
20%
40%
60%
80%
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%
20%
40%
60%
80%
100%
0 20 40 60 80 100 120
Rel
ativ
e ab
unda
nce
Time (min)
TY-ERK2 pTpY-ERK2 pY-ERK2 pT-ERK2
-40.0%
-20.0%
0.0%
20.0%
40.0%
60.0%
0 2 4 6 8 10
Rel
ativ
e ab
unda
nce
diffe
renc
e
EGF (ng/mL)
TY-ERK2 pTpY-ERK2 pY-ERK2 pT-ERK2
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)