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
48
Embed
First insights into bacterial Ser/Thr/Tyr phosphoproteome
Microbial Genomics and Secondary Metabolites MedILS, Split, Croatia June 29, 2007. First insights into bacterial Ser/Thr/Tyr phosphoproteome. Boris Ma č ek Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried, Germany. Our workflow: „GeLC-MS“. - PowerPoint PPT Presentation
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
First insights into bacterial Ser/Thr/Tyrphosphoproteome
Boris MačekDepartment of Proteomics and Signal Transduction
Max Planck Institute of BiochemistryMartinsried, Germany
Microbial Genomics and Secondary MetabolitesMedILS, Split, Croatia
June 29, 2007
Aebersold R, Mann M. 2003. Nature 422: 198-207
Our workflow: „GeLC-MS“Our workflow: „GeLC-MS“
High-resolution, accurate, fast scanning MS: FT-MS
Hybrid linear ion trap FT-MS instruments
LTQ-FTICR MS
zm
k
/Non-destructive
Detection:
Electrostatic field: Electromagnetic field:
m/z
B101.535611
m2
Bq
2
7
r
vf c
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., MCP2005 Olsen 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 1396.7813 Da
Instrument LCQ (ion trap)
LTQ (ion trap)
Q-TOF LTQ-FT LTQ-FT (SIM)
Mass accuracy [ppm]
1000
300
50
10
2
Mass accuracy [Dalton]
+/- 1.4
+/- 0.42
+/- 0.07
+/- 0.014
+/- 0.0028
# of tryptic peptides for
m/z 1396.7813
960
344
202
26
11
Quantitation with Stable Isotope Labeling
Element Stable Isotope
1H 2H
12C 13C
14N 15N
16O 18O
Unlabeled peptide:
Labeled peptide:
Quantitation and identification by MS(nanoscale LC-MS/MS)
Arg-12C6 Arg-13C6
Resting cells Treated (drug, GF)
Combine and lyse,protein purification
or fractionation Background protein. Peptide ratio 1:1
Arg-12C6
Arg-13C6
Upregulated protein. Peptide ratio >1
m/z
Arg-12C6
Arg-13C6
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
• 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 bacteriaTwo-component system
Protein phosphorylation in bacteriaPhosphoenolpyruvate:carbohydrate phosphotransferase system (PTS)
Overview of Ser/Thr/Tyr phosphorylationin 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. 2006. Mol Cell Proteomics 6(4): 697-707
Ser/Thr/Tyr phosphorylation in B. subtilis
# of genes
Expressed
Previous studies
P-proteins P-sites
Bacillus subtilis 168* 4100
60% (log)
13 16
# of genes
Expressed
Previous studies This study
P-proteins P-sites P-proteins P-sites
Bacillus subtilis 168* 4100
60% (log)
13 16 78 78
*Macek et al. 2006. Mol Cell Proteomics 6(4): 697-707
Ser/Thr/Tyr phosphorylation in B. subtilis
# of genes
Expressed
Previous studies
P-proteins P-sites
Bacillus subtilis 168* 4100
60% (log)
13 16
y11
y*18++
y16++
y7
y13++
y*19+++
y*18+++
y*17+++
200 300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
740.427
917.504635.683
573.662
825.480
234.145406.229
1114.647
305.182 946.560
1017.598
y5
y14+++
y4
y2
y3
y14++
y*17++
V T A D pS G I H A R P A T V L V Q T A S K
y2y3y4y5y6y7y11y13y14y*17y*18y*19
Hpr protein
Orbitrap full scanC-trap MS/MS (HCD) Precursor m=0.91ppmFragment m<2ppm
TCA CYCLECitrate 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 studies This study
P-proteins P-sites P-proteins P-sites
Bacillus subtilis 168* 4100
60% (log)
13 16 78 78
Escherichia coli K12** 4289
87% (log)
20 12 79 81
Lactococcus lactis 2250
? (log)
1 1 52 68
Halobacterium salinarum 2605
~80% (stat)
1 1 18 15
Overview of prokaryotes studied so far
Genome size (ORFs)
No. of phospho-proteins
No. of detected phosphorylation events
pS (%)
pT (%)
pY (%)
Essential genes (%)
Essential phospho-proteins (%)
E. coli ~4300 79 105 67.9 23.5 8.6 17 >27 B. subtilis ~4100 78 103 69.2 20.5 10.3 6.6 15.4
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. 2007. submitted
0
10
20
30
40
50
60
70
bacteria eukaryotes archaea
%
phosphoproteome proteome
0
10
20
30
40
50
60
bacteria eukaryotes archaea
%
phosphoproteome proteome
Evolutionary conservation of bacterial S/T/Y phosphoproteins
E. coli phosphoproteome B. 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
Conservation of phosphoserine - E. coli
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Bacteria Eukaryotes Archaea
%
pS
non-pS
Conservation of phosphoserines - B. subtilis
0
5
10
15
20
25
30
35
40
45
50
Bacteria Eukaryotes Archaea%
pS
non-pS
Evolutionary conservation of bacterial S/T/Y phosphorylation sites
→ phosphoserine:
Conservation of phosphothreonine - B. subtilis
0
10
20
30
40
50
60
70
Bacteria Eukaryotes Archaea
%
pT
non-pT
Evolutionary conservation of bacterial S/T/Y phosphorylation sites
→ phosphothreonine:
Bacterial S/T/Y phosphoproteins with P-sitesconserved from Archaea to H. sapiens