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First insights into bacterial Ser/Thr/Tyr phosphoproteome Boris Maček Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry.

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Presentation on theme: "First insights into bacterial Ser/Thr/Tyr phosphoproteome Boris Maček Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry."— Presentation transcript:

1 First insights into bacterial Ser/Thr/Tyr phosphoproteome Boris Maček Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried, Germany Microbial Genomics and Secondary Metabolites MedILS, Split, Croatia June 29, 2007

2 Aebersold R, Mann M Nature 422: Our workflow: „GeLC-MS“

3 High-resolution, accurate, fast scanning MS: FT-MS Hybrid linear ion trap FT-MS instruments LTQ-FTICR MS Non-destructive Detection: Electrostatic field:Electromagnetic field: Parts per million mass accuracy In a 7-Tesla magnetic field an ion with m/z =100 will spin 1,000,000 cycles (travel ~ 30 km) in a 1 sec. observation period Olsen JV et al., MCP2005Olsen JV et al., MCP2004

4 High-mass accuracy – why is it important? Consider all theoretical tryptic peptide masses from the human IPI database (> 40,000 protein sequence entries) Example: Tryptic HSP-70 peptide: ELEEIVQPIISK, mass Da

5 Quantitation with Stable Isotope Labeling ElementStable Isotope 1H1H 2H2H 12 C 13 C 14 N 15 N 16 O 18 O Unlabeled peptide: Labeled peptide:

6 Quantitation and identification by MS (nanoscale LC-MS/MS) Arg- 12 C 6 Arg- 13 C 6 Resting cells Treated (drug, GF) Combine and lyse, protein purification or fractionation Background protein. Peptide ratio 1:1 Arg- 12 C 6 Arg- 13 C 6 Upregulated protein. Peptide ratio >1 m/z Arg- 12 C 6 Arg- 13 C 6 Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) ”normal AA””heavy AA” Proteolysis (trypsin, Lys-C, etc.) Ong SE et. al., Mol Cell Proteomics 2002 Stable isotope dilution: same physico-chemical properties

7 Cell/organism must be auxotrophic for the corresponding AA Growth in defined media lacking the SILAC labeling amino acid (e.g. Arg, Lys) Stable Isotope Labeled Amino Acids: Growth supplements (e.g. dialyzed serum) if necessary SILAC requirements Arg- 13 C6 (Δm=6 Da) Arg- 13 C6 15 N4 (Δm=10 Da) Lys- 13 C6 (Δm=6 Da) Lys- 13 C6 15 N2 (Δm=8 Da)

8 Quantitation Software –

9 Protein Quantitation (Myosin IX)

10 Cell culture days Cell harvest & trypsin digestion Strong cation exchange Chromatography pH<3 ½ - 1 dayO.N. ½ day TiO 2 Chromatography pH<3 (bind) pH>10 (elute) ½ day1-2 days LC-MS pH~1 Data Analysis Gel-free phosphoproteome analysis workflow 1-2 days

11 Larsen et al. (2005) Mol Cell Proteomics 4: Phosphopetide enrichment by Titansphere (TiO 2 ) chromatography Competitive binding of peptides with DHB <<

12 LC separation Proxeon nano-ESI source Agilent 1100, Proxeon nano-HPLC systems self-packed 75 μm x ~10 cm Porous C 18 HPLC columns flow ~250 nL/min

13 Hybrid linear ion trap FTICR MS: LTQ FT (Thermo Scientific)

14 LTQ-FT data-dependent experiments Ion trap MS: + sensitivity (MS/MS mode) and speed  resolution, mass accuracy and dynamic range FTICR MS: + resolution, mass accuracy and dynamic range  sensitivity (MS/MS mode) and speed LTQ-FT: The best from both instruments Two Mass Spectrometers in one - High duty-cycle MS-FullSIM-MS 1stSIM-MS 2ndSIM-MS 3rd MS FT-MS IT-MS LTQ-FT MS/MS optimized scan cycle: Time [msec] Scan typeAGC FT-MS Full5,000,000 FT-MS SIM50,000 IT-MS/MS10,000

15 Phosphopeptide-directed MS 3 Beausoleil SA et al. (2004) PNAS 101:

16 Recent advances in FT-MS: LTQ-Orbitrap (Thermo) Non-destructive Detection: FullSIM1SIM2SIM3 MS FT: LTQ: Time [s] FullSIM1SIM2SIM3 MS Time [s] FullSIM1SIM2SIM3 MS FullSIM1SIM2SIM3 MS FT: LTQ: MS 2 LTQ: Time [s] MS Time [s] MS 2 2 MS 2 Orbitrap: LTQ: Full scan

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19 LTQ-Orbitrap in the analysis of PTMs Multi-stage activation „Hot“ CID

20 CID with Multi-Stage Activation (MSA) m/z 30ms Precursor Da- 49 Da- 98 Da 30ms wb Pseudo MS 3 Easy to identify multiply-phosphorylated peptides: TiO2-enrichment of flow through from SCX (HeLa_EGF_CE_0_5_10) 4, 5 and 6 phosphates

21 Informative low mass ions – reporter ions (Phosphotyrosine immonium ion, m/z = ) CID in the C-trap (”Hot” CID or HCD)

22 Intracellular signaling networks (EGFR, HeLa) (www.phosida.com) Olsen et al. (2006) Cell 127(3): identified more than 2200 phosphoproteins determined more than 6600 phosphorylation sites pS (87%)/pT (12%)/pY (1.5%) less than 15% sites regulated by EGF treatment → systems biology modeling of signaling networks

23 Protein phosphorylation in bacteria Two-component system

24 Protein phosphorylation in bacteria Phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS)

25 Overview of Ser/Thr/Tyr phosphorylation in prokaryotes many putative Ser/Thr/Tyr kinases identified (mostly in silico) 2D gel studies suggest presence of hundred(s) of phosphoproteins However: only about 150 proteins from about 35 species shown to be phosphorylated only about 70 Ser/Thr/Tyr phosphorylation sites identified phosphorylation analysis mostly in vitro! → clear need for in-depth detection and characterization of protein phosphorylation in vivo

26 *Macek et al Mol Cell Proteomics 6(4): Ser/Thr/Tyr phosphorylation in B. subtilis # of genes Expressed Previous studies P-proteinsP-sites Bacillus subtilis 168* % (log) 1316

27 # of genes Expressed Previous studiesThis study P-proteinsP-sitesP-proteinsP-sites Bacillus subtilis 168* % (log) *Macek et al Mol Cell Proteomics 6(4): Ser/Thr/Tyr phosphorylation in B. subtilis # of genes Expressed Previous studies P-proteinsP-sites Bacillus subtilis 168* % (log) 1316

28 V T A D pS G I H A R P A T V L V Q T A S K y2y2 y3y3 y4y4 y5y5 y6y6 y7y7 y 11 y 13 y 14 y* 17 y* 18 y* 19 Hpr protein Orbitrap full scan C-trap MS/MS (HCD) Precursor  m=0.91ppm Fragment  m<2ppm

29 pS V I V N A L R K y3y3 y5y5 y6y6 y7y7 b6b6 b8b8 b4b4 b3b3 b2b2 y8y8 CodY – Global regulator of transcription FT-ICR full scan ion-trap MS/MS (CID) Precursor  m=6.39 ppm Fragment  m<0.5 Da

30 GLYCOLYSIS Enolase (eno) L-lactate dehydrogenase (lctE) Triose phosphate isomerase (tpi) G-3-P dehydrogenase (gap) Pyruvate kinase (pykA) Malate dehydrogenase (citH) Phosphoglycerate mutase (pgm) Glucose-6-phosphate isomerase (pgi) Fructose-bisphosphate aldolase (fbaA) Pyruvate dehydrogenase (pdhB) Phosphoglycerate kinase (pgk) Phosphoglucomutase (ybbT) TCA CYCLE Citrate synthase II (citZ) Succinyl-CoA synthetase (sucC, sucD) Phosphorylation in the main pathways of carbohydrate metabolism (B. subtilis)

31 Is S/T/Y phosphorylation common in bacteria?

32 # of genes Expressed Previous studiesThis study P-proteinsP-sitesP-proteinsP-sites Bacillus subtilis 168* % (log) Escherichia coli K12** % (log) Lactococcus lactis 2250 ? (log) Halobacterium salinarum 2605 ~80% (stat) Overview of prokaryotes studied so far

33 E. coli vs. B. Subtilis phosphoproteome phosphoproteomes similar in: size distribution of S/T/Y phosphorylation classes of phosphorylated proteins increased essentiality *Macek et al submitted

34 Evolutionary conservation of bacterial S/T/Y phosphoproteins E. coli phosphoproteomeB. subtilis phosphoproteome test set of 9 archaeal, 53 bacterial and 8 eukaryotic proteomes look for orthologs of bacterial phosphoproteins (2-directional BLAST; Needle) reported as average % of identified phosphoprotein orthologs in tested species compared to the random protein population

35 Evolutionary conservation of bacterial S/T/Y phosphorylation sites → phosphoserine:

36 Evolutionary conservation of bacterial S/T/Y phosphorylation sites → phosphothreonine:

37 Bacterial S/T/Y phosphoproteins with P-sites conserved from Archaea to H. sapiens cysteinyl t-RNA synthetase phosphoglucomutase nucleoside diphosphate kinase pyruvate kinase enolase predicted GTP-binding protein D-3 phosphoglycerate dehydrogenase phosphoglucosamine mutase elongation factor Ef-Tu

38 → mutases are good internal standards for “quality control”!

39 Is S/T/Y phosphorylation a dynamic process?

40 treated Peptide ratio >1 - Downregulation. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC): Bacillus subtilis (Arg -, Lys - ) control nanoLC-MS/MS (Quantitation and identification by MS) Lys- 12 C 6 14 N 2 Treated cells (succinate or low P) Control cells Combine and lyse ”normal AA””heavy AA” (+8Da) Proteolysis (trypsin) Strong cation exchange chromatography(SCX) Titanium oxide chromatography GeLC-MS Lys- 13 C 6 15 N 2 Peptide ratio 1:1 - No change. m/z

41 Dynamics of protein expression in B. subtilis : Growth on succinate

42 Dynamics of protein phosphorylation in B. subtilis : Growth on succinate

43 Ser46: pSIMGVMSLGIAK Ser12: VTADpSGIHARPATVLVQTASK Growth on low succinate: Hpr protein COOHNH 2 S 12 H 15 S 46 GAEITISASGADENDALNALEETMK

44 Dynamics of protein expression in B. subtilis : Growth under low PO 4 3-

45 Dynamics of protein phosphorylation in B. subtilis : Growth under low PO 4 3-

46 Ser46: pSIMGVMSLGIAK Ser12: VTADpSGIHARPATVLVQTASK YDADVNLEYNGK Growth on low PO 4 3- : Hpr protein COOHNH 2 S 12 H 15 S 46

47 Conclusions SCX + TiO 2 + FT MS - a powerful and generic strategy for phosphopeptide enrichment and detection bacteria posess an elaborate Ser/Thr/Tyr phosphoproteome majority of enzymes in the main pathways of carbohydrate metabolism are phosphorylated enzymes of the PTS system are phosphorylated on Ser/Thr/Tyr → possible cross-talk Ser/Thr/Tyr phosphorylation is dynamic process → likely regulatory role phosphoroteins and phosphorylation sites show increased evolutionary conservation at least 9 P-sites conserved from Archaea to man: ancient regulatory role?

48 Acknowledgements Max-Planck-Institute for Biochemistry Matthias Mann Florian Gnad Jesper V. Olsen Chanchal Kumar Technical University of Denmark Ivan Mijakovic Boumediene Soufi Dina Petranovic Thermo Scientific Stevan Horning Oliver Lange


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