Protein phosphorylation – identification and new technologies for quantitative analysis
1. Detection, identification, and mapping of phosphoproteins
2. New methods for quantitative analysis of protein phosphorylation by mass spectrometry
1. SILAC
2. AQUA peptides
3. Monitoring protein phosphorylation by Bio-Plex
Summary of phosphoprotein and phosphorylation site identification
Cell lineNumber of
phosphoproteins
Number of phosphorylation
sites
WEHI 231 120 198
RAW 264.7 265 225
1. Shu H, Chen S, DeCamp D, Hsueh RC, Mumby M, Brekken D. Identification of the In Vivo Phosphorylation Sites in Murine Leukocyte-Specific Protein 1. AfCS Research Reports [online]. 2003, Vol. 1, no. 8 [cited May 14, 2003].
2. Shu H, Chen S, Bi Q, Mumby M, Brekken D. Identification of WEHI-231 Phosphoproteins and Phosphorylation Sites Using IMAC and LC-MS/MS. AfCS Brief Communications [online], cited February 10, 2004.
3. Shu H, Chen S, Lyons K, Hsueh R, Brekken D. Identification of Immuno-Affinity Isolated Phosphotyrosine Proteins from WEHI-231 Cells. AfCS Brief Communications [online], cited March 21, 2003.
4. Shu H, Chen S, Bi Q, Mumby M, Brekken DL. Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line. Mol Cell Proteomics 3:279-286, 2004.
5. Brekken D, Bi Q, Lyons K, Sethuraman D, Mumby M, Shu H. A Database of Phosphoproteins and Phosphorylation Sites in the Murine RAW 264.7 Macrophage Cell Line. in preparation
Some phosphotyrosine proteins identified in pervanadate-treated RAW cells
Btk Dok1
Fgr Dok2
Lck PLC2
Lyn SHIP
FcεRI Shp1
Fyn Shp2
Fyb/SLAP130 SHPS-1
Hematopoietic cell specific Lyn substrate/HS1
SLP-76
Immunoglobulin superfamily member 4
Identification of PKA substrates in RAW cells
Stimulate RAW with8-Br-cAMP + CL-A
Immunoisolation with anti-PKA substrates antibodies (CST)
Trypsin1D gel
Trypsin
LC-MS/MS(protein ID)
IMAC
LC-MS/MS(protein & site ID)
Ctr
l
8-B
r+ C
L-A
Lamin A/C
NuMA
• phosphopeptides identified by loss of phosphate during CID (MS/MS)
• all spectra confirmed by manual inspection
Phosphoproteins identified in RAW cells treated with 8-Br-cAMP
Protein namePeptide sequence(peptide ID’d is underlined)
Contains predicted PKA site
Acyl-Coenzyme A binding domain containing 4
PQPLKQRS*PRRTR Yes (T103)
DNA methyltransferase MmuI KLESHT*VPVQSR Yes (S254, T324)
DNA methyltransferase MmuI VPALAS*PAGSLPDHVR Yes (S254, T324)
Heterogeneous nuclear ribonucleoprotein U
AAGKSS*GPTSLFAVTVAPPGAR Yes (S26)
Lamin A RSFRS*VGGSGGGSFGDNLVTR Yes (T10)
Lamin A/C LRLS*PSPT*SQR Yes1 (T45, S278)
Lamin C SGAQASSTPLS*PTRITRL Yes (T10)
Mitochondrial Solute Carrier protein RDFY*WLR No
Nucleolin ALVPT*PGKK Yes (T57)
Raly protein (an hnRNP) GRLS*PVPVPR Yes (S119, T135)
Ribosomal protein L7 VATVPGT*LKKKVP No
Vasodilator-stimulated phosphoprotein (VASP)
KLRKVS*KQEEASGGPLAPK Yes1 (S157, S239, T274)
1known PKA substrate
Synergistic phosphorylation of VASP (S157) in response to isoproterenol plus sphingosine-1-phosphate
Iso+S1PControl Isoproterenol
VASP
Sphingosine-1-P
1 2 4 6 10 20
RhoGDI
1 2 4 6 10 20 1 2 4 6 10 20 1 2 4 6 10 20 Time (min)
Time (minutes)
VA
SP
S15
7 P
hosp
hory
latio
n (
fold
cha
nge)
Changes in cAMP levels and VASP phosphorylation in response to isoproterenol plus sphingosine-1-phoshate
0
5
10
15
20
25
30
0 5 10 15 20
0
2
4
6
8
10
cAMPp-VASP
Ch
ang
e in
cA
MP
(fo
ld)
Time (min)
Ch
ang
e in
VA
SP
pho
spho
rylaito
n (fold
)
P
Vinculin
VASP is a member of the Ena/VASP family of adapter proteins
Fyb/SLAP Lyn
P
EVH1 Pro-rich EVH2
S157 S239
PKA
FocalAdhesions
FcR signalingPhagocytosis
WASP
Arp2/3
Actinnucleation
FcR signalingPhagocytosis
Actin binding
New methods for quantitative analysis of phosphorylation of FXM proteins
• Phosphopeptides are usually difficult to detect by mass spectrometry
• To “hedge our bets”, characterization and validation experiments have utilized RAW cells expressing tagged-FXM proteins
• Stable RAW cell populations expressing FLAG-tagged FXM proteins produced via retrovirus transduction and drug selection
• Stable cell populations treated with ligands or phosphatase inhibitors
• Proteins immunoprecipitated with anti-FLAG antibody
• Analyzed by immunoblotting and mass spectrometryFlag-Akt1Grb2-FlagFTM-Erk1Flag-Grk2Flag-SHP2Flag-BtkFlag-Syk
Expression, phosphorylation, and immunoprecipitation of tagged FXM proteins in RAW cells
Detection of Erk1 phosphopeptide by mass spectrometry
Mass spectrum of FLAG-Erk1 tryptic peptides(negative ion mode)
zoom in
Detection of the Erk1 phosphopeptides in RAW cells stimulated with LPS
singly phosphorylated peptide3-
doubly phosphorylated peptide3-
IADPEHDHTGFLT*EY*VATR
Quantitation of protein phosphorylation - the SILAC method
A quantitative proteomic method
Global quantitation of changes in protein abundance
Detection of biomarkers
Specific enrichment of proteins in IPs or affinity capture expts
Quantitation of changes in protein phosphorylation
SILAC – detection of “light” and “heavy” peptide pairs by nanospray mass spectrometry
light
heavy
SILAC validation in RAW cellstime course of 13C-arginine incorporation
Day 1 Day 2 Day 3Day 0
1. RAW 264.7 cells switched into medium containing 13C6-arginine on day 0
2. Lysates resolved by SDS-PAGE
3. Prominent protein band at 90 kDa (Hsp90) excised from each lane and digested with trypsin
4. Peptides analyzed by MS to detect heavy and light peptide pairs
Hsp90
50 kDa
SILAC validation in RAW cellsresults of time course experiments
day 3heavy
lightlight
day 2
lightlight heavy day 1day 0
755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z
2
4
6
8
10
12
14
16
18757.6151
758.1204
758.6155
755.5783 760.5935 761.5872759.5942 764.5780756.5713 762.5709759.1287 763.5749
755.0874 764.1196756.0558
755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z
2468
101214161820222426283032
760.6151
757.6045
761.1203758.1092
761.6098758.6022
755.5810 764.5780763.5638
759.0533 762.0913 764.0617756.5557 759.5726755.0444
755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0
m/z
2
4
6
8
10
12
14
16
18
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22
24
760.6201
761.1215
761.6160
757.6033
758.1087 764.0547758.5869755.5809 762.0987765.0367763.5799
756.5613 759.5730 760.0842 763.0434759.0714
755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z
5
10
15
20
25
30
35
40
Ion
Inte
nsity
(co
unts
)
760.6319
761.1297
761.6246
762.1236757.5946 763.5809755.5774 764.5798758.5864756.5751 759.5683 760.1281
763.0774756.0610
heavy
Ion
Inte
nsity
(co
unts
)
Mass spectra of light and heavy pairs of Hsp90 peptide (ADHGEPIGR*)
SILAC validation in RAW cellsaccurate quantitation of peptides by mass spectrometry
Absolute quantitation of protein phosphorylation – the AQUA peptide method
• Another quantitative proteomic method
• Allows absolute quantitation of the amount of protein
• Allows quantitation of the stoichiometry of protein phosphorylation
• Universal application to any protein or phosphoprotein
• Quantitation relies on internal standards comprised of synthetic peptides containing an isotopically-labeled amino acid
AQUA peptide design for FLAG-Akt1 (Ser473)
MDYKDDDDKGAGAGSSSGHQTSLYKKAGSTMNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA
Ser473 tryptic peptide
FLAG + attB1 sequence
endogenous phosphopeptideRPHFPQFS*YSASGTA + PO3
m/z of M2- = 865
synthetic “AQUA” phosphopeptide RPHFPQF(13C9
15N1)S*YSASGTA + PO3
m/z of M2- = 870
AQUA method – mass spectrum of FLAG-Akt1 from calyculin-A treated RAW cells
Zoom in on this area
AQUA peptide – FLAG-Akt1 (Ser473) zoomed scan
Endogenous phosphopeptide
13C,15N-labeledAQUA phosphopeptide
Planned uses of SILAC and AQUA methods
• SILAC
– in a targeted way to quantitate phosphorylation of specific proteins (e.g., FXM)
– in a global way to identify known and novel proteins whose phosphorylation is altered by ligands/perturbations – coupled with antibody and IMAC enrichment methods
• AQUA
– in a targeted manner to quantitate phosphorylation of FXM proteins where a suitable phospho-specific antibody is not available
– quantitate the absolute amounts of phosphoproteins and the stoichiometry of phosphorylation in response to stimuli/perturbations
• Current efforts
– finish implementing both methods
– increase sensitivity – selected reaction monitoring (20X); new instrumentation (50X)
The AfCS Protein Chemistry Lab
Deirdre BrekkenLead Scientist
Hongjun ShuLead Scientist
Farah El Mazouni Kathy Lyons Deepa Sethuraman Robert Cox Qun Bi Laura Draper
Liquid suspension array for sandwich immunoassay of protein phosphorylation
ERK
Akt
P-ERK
P-Akt
Spectrally distinct fluorescent beads (100), conjugated to conventional antibodies directed against target proteins, are incubated with cell lysate
Phospho and non-phospho ERK (or Akt) are captured on beads
The immuno-complex is labeled with streptavidin-PE and fluorescence of both PE and beads are quantified with Bio-Plex system
The protein-antibody complexes are incubated with biotinylated antibodies specific for phosphorylated ERK and Akt
P-STAT 3
0
2
4
6
8
10
Fold
Cha
nge
IL10 - 5 min IL10 - 20 min
EWC040305B
Bio-PlexEC50: ~0.1 nM
Western
P-STAT3
0
2
4
6
8
10
12
14F
old
Ch
ang
eIL10 - 5 min IL10 - 20 min
EWC040305B
EC50: ~0.1 nM
P-STAT3
0’ 5 min IL-10 20 min IL-10
Western Image
Dose-response of STAT3 phosphorylation to IL-10
Same EC 50 estimated by Western and Bio-Plex
Heping HanAntibody Lab
Bio-Plex
Western
MCF P2C P2C+MCF
0’ 1’ 3’ 10’ 30’ 1’ 3’ 10’ 30’ 1’ 3’ 10’ 30’
P-ERKs
Western Image
P- ERK1/2
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1
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7
0 5 10 15 20 25 30 35
Time (min)
Fo
ld C
han
ge
200pM MCF
350nM P2C
350nM P2C, 200pM MCF
P- ERK1
0.00.51.01.52.02.53.03.54.04.5
0 10 20 30 40
Stimulation Time (min.)
Fo
ld C
ha
ng
e
MCF (200 pM)
P2C (350 nM)
P2C + MCF
EWC040305CP- ERK2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 5 10 15 20 25 30 35
Stimulation Time (min.)
Fo
ld C
ha
ng
e
MCF (200 pM)
P2C (350 nM)
P2C + MCF
EWC040305C
Time course patterns of single and Double ligand-stimulated phosphorylation of ERKs are identical by Western and Bio-Plex
Erk phosphorylation in response to single and double ligands
Heping HanAntibody Lab
Conclusions:
• Bio-Plex results are VERY similar to blotting results
• Bio-Plex has the potential to triple or quadruple the number of phosphoproteins currently monitored
• Bio-Plex has the potential to massively increase throughput in screening phosphoproteins
• More sets of antibodies for phosphoproteins are needed to help AfCS (currently 11)
P-Stat 3
02468
10
Untreat IL10 15' LPS 30' Untreat IL10 15' LPS 30'
Western Bio-Plex
P-ATF2
0
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15
18
Untreat IL10 15' LPS 30' Untreat IL10 15' LPS 30'
Fo
ld C
han
ge
Westren Bio-Plex
P-ERKs
0
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10
Untreat IL10 15' LPS 30' Untreat IL10 15' LPS 30'
Fo
ld C
han
ge
Western Bio-Plex
Summary comparison of Bio-Plex and Western Blotting for quantifyingligand-induced phosphorylation of ATF-2, ERKs, and STAT 3
Validation of the Bio-Plex assay for protein phosphorylation is an ongoing collaboration between
The AfCS Antibody Lab
and
Bio-Rad Laboratories
and
Cell Signaling Technology
Bio-Plex