first insights into bacterial ser/thr/tyr phosphoproteome
DESCRIPTION
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 PresentationTRANSCRIPT
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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
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Aebersold R, Mann M. 2003. Nature 422: 198-207
Our workflow: „GeLC-MS“Our workflow: „GeLC-MS“
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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
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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
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Quantitation with Stable Isotope Labeling
Element Stable Isotope
1H 2H
12C 13C
14N 15N
16O 18O
Unlabeled peptide:
Labeled peptide:
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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
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• 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
C
NH2
CH
CH2
H2C
CH2
HN
C
NH
H2N
O
OH
L-arginine
C
H2N
CH
CH2H2C
CH2H2C
H2N O
OH
L-lysine
Arg-13C6 (Δm=6 Da)Arg-13C615N4 (Δm=10 Da)
Lys-13C6 (Δm=6 Da)Lys-13C615N2 (Δm=8 Da)
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Quantitation Software – http://msquant.sourceforge.net
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No. Peptide Sequence Average Peptide Ratio1 GILTPR 1.065±4.92%*2 WLVLR 1.048±5.58%3 NCAAYLR 0.999±4.54%4 NTNPNFVR 1.068±4.36%5 GALALEEKR 1.055±5.80%6 GDLPFVVTR 1.084±6.47%7 ALELDSNLYR 1.024±4.91%8 AGVLAHLEEER 1.073±2.33%9 LDPHLVLDQLR 0.992±2.61%
10 VSHLLGINVTDFTR 0.954±5.51%11 AGKLDPHLVLDQLR 1.073±2.68%12 KQELEEICHDLEAR 1.040±4.44%
1.040.04
3.82%* Relative standard deviation
Average Protein Abundance RatioSD
RSD (%)
Protein Quantitation (Myosin IX)
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Cell culture
days
Cell harvest & trypsin digestion
Strong cationexchange
ChromatographypH<3
½ - 1 day O.N. ½ day
TiO2
ChromatographypH<3 (bind)
pH>10 (elute)
½ day 1-2 days
LC-MSpH~1
DataAnalysis
Gel-free phosphoproteome analysis workflow
1-2 days
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Larsen et al. (2005) Mol Cell Proteomics 4:873-886
Phosphopetide enrichment by Titansphere (TiO2) chromatography
Competitive binding of peptides with DHB
< <
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LC separation
• Proxeon nano-ESI source • Agilent 1100, Proxeon nano-HPLC systems• self-packed 75 μm x ~10 cm Porous C18 HPLC columns• flow ~250 nL/min
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Hybrid linear ion trap FTICR MS:LTQ FT (Thermo Scientific)
m/z
B101.535611
m2
Bq
2
7
r
vf c
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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-Full SIM-MS 1st SIM-MS 2nd SIM-MS 3rd
MS2 MS2 MS2
0 300 600 900 1200 1500 1800
FT-MS
IT-MS
LTQ-FT MS/MS optimized scan cycle:
Time [msec]
Scan type AGC
FT-MS Full 5,000,000
FT-MS SIM 50,000
IT-MS/MS 10,000
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Phosphopeptide-directed MS3
Beausoleil SA et al. (2004) PNAS 101:12130-35.
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Recent advances in FT-MS: LTQ-Orbitrap (Thermo)
zm
k
/
Non-destructive Detection:
Full SIM1 SIM2 SIM3
MS2 MS3 MS2 MS3 MS2 MS3
FT:
LTQ:
0 1 2Time [s]
Full SIM1 SIM2 SIM3
MS2 MS3 MS2 MS3 MS2 MS3
0 1 2Time [s]
Full SIM1 SIM2 SIM3
MS2 MS3 MS2 MS3 MS2 MS3
Full SIM1 SIM2 SIM3
MS2 MS3 MS2 MS3 MS2 MS3
FT:
LTQ: MS2 MS MS MS MSLTQ:
0 1 2Time [s]
MS2 MS MS MS MS
0 1 2Time [s]
MS2 MS MS MS MSMS2 MS2 MS2 MS2 MS2
Orbitrap:
LTQ:
Full scan
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LTQ-Orbitrap in the analysis of PTMs
Multi-stage activation
„Hot“ CID
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CID with Multi-Stage Activation (MSA)
m/z
30ms
Precursor - 32.6 Da - 49 Da - 98 Da
30ms 30ms 30ms
wbPseudo MS3
Easy to identify multiply-phosphorylated peptides:
TiO2-enrichment of flow through from SCX (HeLa_EGF_CE_0_5_10)
4, 5 and 6 phosphates
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Informative low mass ions – reporter ions(Phosphotyrosine immonium ion, m/z = 216.0426)
CID in the C-trap (”Hot” CID or HCD)
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Intracellular signaling networks (EGFR, HeLa)
(www.phosida.com)
Olsen et al. (2006) Cell 127(3):635-648
• 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
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Protein phosphorylation in bacteriaTwo-component system
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Protein phosphorylation in bacteriaPhosphoenolpyruvate:carbohydrate phosphotransferase system (PTS)
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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
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*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
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# 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
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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
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200 300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
491.36
505.00283.00700.27482.73 601.27407.27 643.27350.55254.27 566.18 740.18 836.36213.09 771.73 873.55186.82 936.73 982.09 1022.18 1062.18
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
601.31 700.30282.13
254.20407.44
683.42572.31 835.35613.40416.39 456.61 566.43712.48 813.50
381.19185.10 301.41 854.58461.97 667.45 769.45213.15169.10 499.42 877.74
y3
y5 y6
y7
-H3PO4
MS3
[M+2H]2+
540.2988
b8b6y8++
y7++
y6++
b3
b2
b4
200 300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
491.36
505.00283.00700.27482.73 601.27407.27 643.27350.55254.27 566.18 740.18 836.36213.09 771.73 873.55186.82 936.73 982.09 1022.18 1062.18
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
601.31 700.30282.13
254.20407.44
683.42572.31 835.35613.40416.39 456.61 566.43712.48 813.50
381.19185.10 301.41 854.58461.97 667.45 769.45213.15169.10 499.42 877.74
200 300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
491.36
505.00283.00700.27482.73 601.27407.27 643.27350.55254.27 566.18 740.18 836.36213.09 771.73 873.55186.82 936.73 982.09 1022.18 1062.18
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
601.31 700.30282.13
254.20407.44
683.42572.31 835.35613.40416.39 456.61 566.43712.48 813.50
381.19185.10 301.41 854.58461.97 667.45 769.45213.15169.10 499.42 877.74
y3
y5 y6
y7
-H3PO4
MS3
[M+2H]2+
540.2988
b8b6y8++
y7++
y6++
b3
b2
b4
pS V I V N A L R Ky3y5y6y7
b6 b8b4b3b2
y8
CodY – Global regulator of transcription
FT-ICR full scanion-trap MS/MS (CID)Precursor m=6.39 ppmFragment m<0.5 Da
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GLYCOLYSISEnolase (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 CYCLECitrate synthase II (citZ)Succinyl-CoA synthetase (sucC, sucD)
Phosphorylation in the main pathways of carbohydrate metabolism (B. subtilis)
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Is S/T/Y phosphorylation common in bacteria?
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# 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
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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
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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
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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:
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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:
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Bacterial S/T/Y phosphoproteins with P-sitesconserved 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
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→ mutases are good internal standards for “quality control”!
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Is S/T/Y phosphorylation a dynamic process?
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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-12C6
14N2
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-13C615N2
Peptide ratio 1:1 - No change.
m/z
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-10-9-8-7-6-5-4-3-2-10123456789
10
0 100 200 300 400 500 600 700log(
2)
6-phospho-beta-glucosidase
similar to phosphomannomutasebeta-glucosidase
glucose kinasePTS glucose-specific enzyme II
argininosuccinate synthasemethionyl-tRNA synthetase
transcriptional regulator CodYDNA polymerase III
succinyl-CoA synthetase
transcriptional regulator GutRsimilar to phosphoglucomutase
-10-9-8-7-6-5-4-3-2-10123456789
10
0 100 200 300 400 500 600 700log(
2)
6-phospho-beta-glucosidase
similar to phosphomannomutasebeta-glucosidase
glucose kinasePTS glucose-specific enzyme II
argininosuccinate synthasemethionyl-tRNA synthetase
transcriptional regulator CodYDNA polymerase III
succinyl-CoA synthetase
transcriptional regulator GutRsimilar to phosphoglucomutase
Dynamics of protein expression in B. subtilis :Growth on succinate
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-2
-1
0
1
2
3
4
log
(2)
protein
phospho
protein 0.321 0.538 3.489 2.466 -0.19 0.072 0.469 0.246 0.225 0.225
phospho 0.6 0.407 2.593 1.059 0.804 -0.15 -0.43 -0.59 -0.32 -1.22
ybbT rsbW yerA ispU yvcT rocA fbaA rocDptsH (S12)
ptsH (S46)
Dynamics of protein phosphorylation in B. subtilis :Growth on succinate
![Page 43: First insights into bacterial Ser/Thr/Tyr phosphoproteome](https://reader035.vdocument.in/reader035/viewer/2022062305/56815731550346895dc4d147/html5/thumbnails/43.jpg)
Ser46: pSIMGVMSLGIAK
Ser12: VTADpSGIHARPATVLVQTASK
Growth on low succinate: Hpr protein
COOHNH2
S12 H15 S46
GAEITISASGADENDALNALEETMK
GAEITISASGADENDALNALEETMK
![Page 44: First insights into bacterial Ser/Thr/Tyr phosphoproteome](https://reader035.vdocument.in/reader035/viewer/2022062305/56815731550346895dc4d147/html5/thumbnails/44.jpg)
-10-9-8-7-6-5-4-3-2-10123456789
10
0 100 200 300 400 500 600 700 800 900log(
2)
PTS sucrose-specific enzyme IIalkaline phosphatase A
inositol-monophosphate dehydrogenasecarbon starvation-induced protein
ATP synthaseGroEL
DNA polymerase IIIcysteine synthase
PTS enzyme I
-10-9-8-7-6-5-4-3-2-10123456789
10
0 100 200 300 400 500 600 700 800 900log(
2)
PTS sucrose-specific enzyme IIalkaline phosphatase A
inositol-monophosphate dehydrogenasecarbon starvation-induced protein
ATP synthaseGroEL
DNA polymerase IIIcysteine synthase
PTS enzyme I
Dynamics of protein expression in B. subtilis :Growth under low PO4
3-
![Page 45: First insights into bacterial Ser/Thr/Tyr phosphoproteome](https://reader035.vdocument.in/reader035/viewer/2022062305/56815731550346895dc4d147/html5/thumbnails/45.jpg)
-8.00
-6.00
-4.00
-2.00
0.00
2.00
4.00
log
(2) Protein
Phospho
Protein -3.06 -3.97 0.53 0.40 0.40 0.29 1.31 -0.49 -4.15 -5.33 -6.43 -4.27 -4.99
Phospho -3.05 -3.22 0.47 0.36 1.76 2.19 2.65 2.76 0.88 -2.67 -1.91 -3.37 -1.60
ybbT ypfD sodAptsH (S46)
ptsH (S12)
yfkK ypsB yvaB tpi yvcT fbaA pta rocA
Dynamics of protein phosphorylation in B. subtilis :Growth under low PO4
3-
![Page 46: First insights into bacterial Ser/Thr/Tyr phosphoproteome](https://reader035.vdocument.in/reader035/viewer/2022062305/56815731550346895dc4d147/html5/thumbnails/46.jpg)
Ser46: pSIMGVMSLGIAK
Ser12: VTADpSGIHARPATVLVQTASK
YDADVNLEYNGK
YDADVNLEYNGK
Growth on low PO43-: Hpr protein
COOHNH2
S12 H15 S46
![Page 47: First insights into bacterial Ser/Thr/Tyr phosphoproteome](https://reader035.vdocument.in/reader035/viewer/2022062305/56815731550346895dc4d147/html5/thumbnails/47.jpg)
Conclusions
• SCX + TiO2 + 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?
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Acknowledgements
Max-Planck-Institute for BiochemistryMatthias MannFlorian GnadJesper V. OlsenChanchal Kumar
Technical University of DenmarkIvan MijakovicBoumediene SoufiDina Petranovic
Thermo ScientificStevan HorningOliver Lange