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
Aebersold R, Mann M Nature 422: Our workflow: „GeLC-MS“
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
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
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:
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
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)
Quantitation Software –
Protein Quantitation (Myosin IX)
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
Larsen et al. (2005) Mol Cell Proteomics 4: Phosphopetide enrichment by Titansphere (TiO 2 ) chromatography Competitive binding of peptides with DHB <<
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
Hybrid linear ion trap FTICR MS: LTQ FT (Thermo Scientific)
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
Phosphopeptide-directed MS 3 Beausoleil SA et al. (2004) PNAS 101:
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
LTQ-Orbitrap in the analysis of PTMs Multi-stage activation „Hot“ CID
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
Informative low mass ions – reporter ions (Phosphotyrosine immonium ion, m/z = ) CID in the C-trap (”Hot” CID or HCD)
Intracellular signaling networks (EGFR, HeLa) ( 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
Protein phosphorylation in bacteria Two-component system
Protein phosphorylation in bacteria Phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS)
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
*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
# 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
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
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
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)
Is S/T/Y phosphorylation common in bacteria?
# 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
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
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
Evolutionary conservation of bacterial S/T/Y phosphorylation sites → phosphoserine:
Evolutionary conservation of bacterial S/T/Y phosphorylation sites → phosphothreonine:
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
→ mutases are good internal standards for “quality control”!
Is S/T/Y phosphorylation a dynamic process?
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
Dynamics of protein expression in B. subtilis : Growth on succinate
Dynamics of protein phosphorylation in B. subtilis : Growth on succinate
Ser46: pSIMGVMSLGIAK Ser12: VTADpSGIHARPATVLVQTASK Growth on low succinate: Hpr protein COOHNH 2 S 12 H 15 S 46 GAEITISASGADENDALNALEETMK
Dynamics of protein expression in B. subtilis : Growth under low PO 4 3-
Dynamics of protein phosphorylation in B. subtilis : Growth under low PO 4 3-
Ser46: pSIMGVMSLGIAK Ser12: VTADpSGIHARPATVLVQTASK YDADVNLEYNGK Growth on low PO 4 3- : Hpr protein COOHNH 2 S 12 H 15 S 46
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?
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