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

20

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

0

1

2

3

4

5

6

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

3

6

9

12

15

18

Untreat IL10 15' LPS 30' Untreat IL10 15' LPS 30'

Fo

ld C

han

ge

Westren Bio-Plex

P-ERKs

0

2

4

6

8

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