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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

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Protein phosphorylation – identification and new technologies for quantitative analysis

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  1. 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

  2. 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

  3. Some phosphotyrosine proteins identified in pervanadate-treated RAW cells

  4. 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)

  5. Phosphoproteins identified in RAW cells treated with 8-Br-cAMP 1known PKA substrate

  6. 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)

  7. 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)

  8. 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

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

  12. singly phosphorylated peptide3- doubly phosphorylated peptide3- Detection of the Erk1 phosphopeptides in RAW cells stimulated with LPS 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 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

  16. 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

  17. SILAC validation in RAW cellsaccurate 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) 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

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

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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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)

  28. 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

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