1. 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

2. Summary of phosphoprotein and phosphorylation site identification Cell line Number of phosphoproteins Number of phosphorylation sites WEHI 231120198 RAW 264.7265225 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

3. Some phosphotyrosine proteins identified in pervanadate-treated RAW cells BtkDok1 FgrDok2 Lck PLC  2 LynSHIP FcεRIShp1 FynShp2 Fyb/SLAP130SHPS-1 Hematopoietic cell specific Lyn substrate/HS1 SLP-76 Immunoglobulin superfamily member 4

4. 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 LC-MS/MS (protein ID) IMAC LC-MS/MS (protein & site ID) Ctrl 8-Br+ CL-A Lamin A/C NuMA phosphopeptides identified by loss of phosphate during CID (MS/MS) all spectra confirmed by manual inspection

5. Phosphoproteins identified in RAW cells treated with 8-Br-cAMP Protein name Peptide sequence (peptide ID’d is underlined) Contains predicted PKA site Acyl-Coenzyme A binding domain containing 4 PQPLKQRS*PRRTRYes (T103) DNA methyltransferase MmuIKLESHT*VPVQSRYes (S254, T324) DNA methyltransferase MmuIVPALAS*PAGSLPDHVRYes (S254, T324) Heterogeneous nuclear ribonucleoprotein U AAGKSS*GPTSLFAVTVAPPGARYes (S26) Lamin ARSFRS*VGGSGGGSFGDNLVTRYes (T10) Lamin A/CLRLS*PSPT*SQRYes 1 (T45, S278) Lamin CSGAQASSTPLS*PTRITRLYes (T10) Mitochondrial Solute Carrier proteinRDFY*WLRNo NucleolinALVPT*PGKKYes (T57) Raly protein (an hnRNP)GRLS*PVPVPRYes (S119, T135) Ribosomal protein L7VATVPGT*LKKKVPNo Vasodilator-stimulated phosphoprotein (VASP) KLRKVS*KQEEASGGPLAPKYes 1 (S157, S239, T274) 1 known PKA substrate

6. Synergistic phosphorylation of VASP (S157) in response to isoproterenol plus sphingosine-1-phosphate Iso+S1PControl Isoproterenol VASP Sphingosine-1-P 12461020 RhoGDI 124610201246102012461020 Time (min) Time (minutes) VASP S157 Phosphorylation (fold change)

7. Changes in cAMP levels and VASP phosphorylation in response to isoproterenol plus sphingosine-1-phoshate Change in cAMP (fold) Time (min) Change in VASP phosphorylaiton (fold)

8. P Vinculin VASP is a member of the Ena/VASP family of adapter proteins Fyb/ SLAP Lyn P EVH1Pro-richEVH2 S157S239 PKA Focal Adhesions FcR signaling Phagocytosis WASP Arp 2/3 Actin nucleation FcR signaling Phagocytosis Actin binding

9. 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

10. Expression, phosphorylation, and immunoprecipitation of tagged FXM proteins in RAW cells

11. Detection of Erk1 phosphopeptide by mass spectrometry Mass spectrum of FLAG-Erk1 tryptic peptides (negative ion mode) zoom in

12. Detection of the Erk1 phosphopeptides in RAW cells stimulated with LPS singly phosphorylated peptide 3- doubly phosphorylated peptide 3- IADPEHDHTGFLT*EY*VATR

13. 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

14. SILAC – detection of “light” and “heavy” peptide pairs by nanospray mass spectrometry light heavy

15. SILAC validation in RAW cells time course of 13 C-arginine incorporation Day 1Day 2Day 3Day 0 1.RAW 264.7 cells switched into medium containing 13 C 6 -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

16. SILAC validation in RAW cells results of time course experiments day 3 heavy light day 2 light heavy day 1day 0 755.0756.0757.0758.0759.0760.0761.0762.0763.0764.0765.0 m/z 2 4 6 8 10 12 14 16 18 757.6151 758.1204 758.6155 755.5783 760.5935 761.5872 759.5942 764.5780 756.5713 762.5709 759.1287 763.5749 755.0874 764.1196 756.0558 755.0756.0757.0758.0759.0760.0761.0762.0763.0764.0765.0 m/z 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 760.6151 757.6045 761.1203 758.1092 761.6098 758.6022 755.5810 764.5780 763.5638 759.0533 762.0913 764.0617756.5557 759.5726 755.0444 755.0756.0757.0758.0759.0760.0761.0762.0763.0764.0765.0 m/z 2 4 6 8 10 12 14 16 18 20 22 24 760.6201 761.1215 761.6160 757.6033 758.1087 764.0547 758.5869 755.5809 762.0987 765.0367 763.5799 756.5613 759.5730 760.0842 763.0434 759.0714 755.0756.0757.0758.0759.0760.0761.0762.0763.0764.0765.0 m/z 5 10 15 20 25 30 35 40 Ion Intensity (counts) 760.6319 761.1297 761.6246 762.1236 757.5946 763.5809 755.5774 764.5798 758.5864 756.5751 759.5683 760.1281 763.0774 756.0610 heavy Ion Intensity (counts) Mass spectra of light and heavy pairs of Hsp90 peptide (ADHGEPIGR*)

17. SILAC validation in RAW cells accurate quantitation of peptides by mass spectrometry

18. 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

19. AQUA peptide design for FLAG-Akt1 (Ser473) MDYKDDDDKGAGAGSSSGHQTSLYKKAGSTMNDVAIVKEGWLHKRGE YIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQCQLMK TERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGLK RQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGK GTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNS RHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARF YGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEG IKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGR LPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLG GGSEDAKEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDE EFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA Ser473 tryptic peptide FLAG + attB1 sequence endogenous phosphopeptide RPHFPQFS*YSASGTA + PO 3 m/z of M 2- = 865 synthetic “AQUA” phosphopeptide RPHFPQF( 13 C 9 15 N 1 )S*YSASGTA + PO 3 m/z of M 2- = 870

20. AQUA method – mass spectrum of FLAG-Akt1 from calyculin-A treated RAW cells Zoom in on this area

21. AQUA peptide – FLAG-Akt1 (Ser473) zoomed scan Endogenous phosphopeptide 13 C, 15 N-labeled AQUA phosphopeptide

22. 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)

23. The AfCS Protein Chemistry Lab Deirdre Brekken Lead Scientist Hongjun Shu Lead Scientist Farah El MazouniKathy LyonsDeepa SethuramanRobert CoxQun Bi Laura Draper

24. 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

25. Bio-Plex EC 50 : ~0.1 nM Western EC 50 : ~0.1 nM P-STAT3 0’5 min IL-1020 min IL-10 Western Image Dose-response of STAT3 phosphorylation to IL-10 Same EC 50 estimated by Western and Bio-Plex Heping Han Antibody Lab

26. Bio-Plex Western MCFP2CP2C+MCF 0’ 1’ 3’ 10’ 30’ P-ERKs Western Image 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 Han Antibody Lab

27. 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) Summary comparison of Bio-Plex and Western Blotting for quantifying ligand-induced phosphorylation of ATF-2, ERKs, and STAT 3

28. 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