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Protein phosphorylation – identification and new technologies for quantitative analysis. Detection, identification, and mapping of phosphoproteins New methods for quantitative analysis of protein phosphorylation by mass spectrometry SILAC AQUA peptides

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Protein phosphorylation – identification and new technologies for quantitative analysis


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protein phosphorylation identification and new technologies for quantitative analysis
Protein phosphorylation – identification and new technologies for quantitative analysis
  • Detection, identification, and mapping of phosphoproteins
  • New methods for quantitative analysis of protein phosphorylation by mass spectrometry
    • SILAC
    • AQUA peptides
  • Monitoring protein phosphorylation by Bio-Plex
summary of phosphoprotein and phosphorylation site identification
Summary of phosphoprotein and phosphorylation site identification
  • 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].
  • 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.
  • 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.
  • 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.
  • 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
Some phosphotyrosine proteins identified in pervanadate-treated RAW cells
identification of pka substrates in raw cells

8-Br+ CL-A

Ctrl

NuMA

  • phosphopeptides identified by loss of phosphate during CID (MS/MS)
  • all spectra confirmed by manual inspection

Lamin A/C

Identification of PKA substrates in RAW cells

Stimulate RAW with

8-Br-cAMP + CL-A

Immunoisolation with anti-PKA substrates antibodies (CST)

Trypsin

1D gel

Trypsin

IMAC

LC-MS/MS

(protein ID)

LC-MS/MS

(protein & site ID)

slide6

Synergistic phosphorylation of VASP (S157) in response to isoproterenol plus sphingosine-1-phosphate

Control

Isoproterenol

Sphingosine-1-P

Iso+S1P

Time (min)

1

2

4

6

10

20

1

2

4

6

10

20

1

2

4

6

10

20

1

2

4

6

10

20

VASP

RhoGDI

VASP S157 Phosphorylation

(fold change)

Time (minutes)

slide7

Changes in cAMP levels and VASP phosphorylation in response to isoproterenol plus sphingosine-1-phoshate

Change in VASP phosphorylaiton (fold)

Change in cAMP (fold)

Time (min)

slide8

Fyb/

SLAP

Lyn

WASP

Vinculin

Arp

2/3

VASP is a member of the Ena/VASP family of adapter proteins

PKA

S157

S239

P

P

EVH1

Pro-rich

EVH2

FcR signaling

Phagocytosis

Focal

Adhesions

Actin binding

FcR signaling

Phagocytosis

Actin

nucleation

new methods for quantitative analysis of phosphorylation of fxm proteins
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 spectrometry

Flag-Akt1

Grb2-Flag

FTM-Erk1

Flag-Grk2

Flag-SHP2

Flag-Btk

Flag-Syk

detection of erk1 phosphopeptide by mass spectrometry

zoom in

Detection of Erk1 phosphopeptide by mass spectrometry

Mass spectrum of FLAG-Erk1 tryptic peptides

(negative ion mode)

detection of the erk1 phosphopeptides in raw cells stimulated with lps

singly phosphorylated peptide3-

doubly phosphorylated peptide3-

Detection of the Erk1 phosphopeptides in RAW cells stimulated with LPS

IADPEHDHTGFLT*EY*VATR

quantitation of protein phosphorylation the silac method
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
SILAC – detection of “light” and “heavy” peptide pairs by nanospray mass spectrometry

light

heavy

silac validation in raw cells time course of 13 c arginine incorporation
SILAC validation in RAW cellstime course of 13C-arginine incorporation

Day 0

Day 1

Day 2

Day 3

  • RAW 264.7 cells switched into medium containing 13C6-arginine on day 0
  • Lysates resolved by SDS-PAGE
  • Prominent protein band at 90 kDa (Hsp90) excised from each lane and digested with trypsin
  • Peptides analyzed by MS to detect heavy and light peptide pairs

Hsp90

50 kDa

silac validation in raw cells results of time course experiments
SILAC validation in RAW cellsresults of time course experiments

Mass spectra of light and heavy pairs of Hsp90 peptide (ADHGEPIGR*)

day 0

day 1

757.6151

760.6151

light

heavy

18

32

light

30

757.6045

16

28

26

14

758.1204

24

761.1203

758.1092

22

12

20

10

18

Ion Intensity (counts)

758.6155

16

761.6098

758.6022

8

14

12

6

10

755.5810

764.5780

755.5783

760.5935

8

761.5872

759.5942

4

763.5638

764.5780

756.5713

762.5709

759.1287

763.5749

759.0533

762.0913

756.5557

764.0617

759.5726

6

755.0444

755.0874

764.1196

4

2

756.0558

2

755.0

756.0

757.0

758.0

759.0

760.0

761.0

762.0

763.0

764.0

765.0

755.0

756.0

757.0

758.0

759.0

760.0

761.0

762.0

763.0

764.0

765.0

m/z

day 2

day 3

m/z

760.6201

760.6319

24

40

heavy

heavy

22

35

20

761.1297

761.1215

18

30

16

25

14

Ion Intensity (counts)

light

12

20

761.6160

10

761.6246

light

15

8

757.6033

6

10

758.1087

764.0547

758.5869

755.5809

762.0987

765.0367

763.5799

762.1236

757.5946

4

763.5809

755.5774

764.5798

756.5613

759.5730

758.5864

760.0842

763.0434

759.0714

756.5751

759.5683

760.1281

5

763.0774

756.0610

2

755.0

756.0

757.0

758.0

759.0

760.0

761.0

762.0

763.0

764.0

765.0

755.0

756.0

757.0

758.0

759.0

760.0

761.0

762.0

763.0

764.0

765.0

m/z

m/z

absolute quantitation of protein phosphorylation the aqua peptide method
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
AQUA peptide design for FLAG-Akt1 (Ser473)

FLAG + attB1 sequence

MDYKDDDDKGAGAGSSSGHQTSLYKKAGSTMNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA

Ser473 tryptic peptide

endogenous phosphopeptide

RPHFPQFS*YSASGTA + PO3

m/z of M2- = 865

synthetic “AQUA” phosphopeptide RPHFPQF(13C915N1)S*YSASGTA + PO3

m/z of M2- = 870

aqua peptide flag akt1 ser473 zoomed scan
AQUA peptide – FLAG-Akt1 (Ser473) zoomed scan

Endogenous

phosphopeptide

13C,15N-labeled

AQUA phosphopeptide

planned uses of silac and aqua methods
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 withantibody 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
The AfCS Protein Chemistry Lab

Deirdre Brekken

Lead Scientist

Hongjun Shu

Lead Scientist

Laura Draper

Farah El Mazouni

Kathy Lyons

Deepa Sethuraman

Robert Cox

Qun Bi

liquid suspension array for sandwich immunoassay of protein phosphorylation

P-ERK

ERK

P-Akt

Akt

Liquid suspension array for sandwich immunoassay of protein phosphorylation

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 protein-antibody complexes are incubated with biotinylated antibodies specific for phosphorylated ERK and Akt

The immuno-complex is labeled with streptavidin-PE and fluorescence of both PE and beads are quantified with Bio-Plex system

dose response of stat3 phosphorylation to il 10
Dose-response of STAT3 phosphorylation to IL-10

Same EC 50 estimated by Western and Bio-Plex

Western Image

P-STAT3

EC50: ~0.1 nM

Western

EC50: ~0.1 nM

Bio-Plex

Heping Han

Antibody Lab

erk phosphorylation in response to single and double ligands
Erk phosphorylation in response to single and double ligands

Western Image

P-ERKs

Western

Time course patterns of single and

Double ligand-stimulated phosphorylation

of ERKs are identical by Western and

Bio-Plex

Bio-Plex

Heping Han

Antibody Lab

slide27

Summary comparison of Bio-Plex and Western Blotting for quantifying

ligand-induced phosphorylation of ATF-2, ERKs, and STAT 3

  • 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)
bio plex
Bio-Plex

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