Download B. subtilis

Microbial Genomics and Secondary Metabolites
MedILS, Split, Croatia
June 29, 2007
First insights into bacterial Ser/Thr/Tyr
phosphoproteome
Boris Maček
Department of Proteomics and Signal Transduction
Max Planck Institute of Biochemistry
Martinsried, Germany
Our workflow: „GeLC-MS“
Aebersold R, Mann M. 2003. Nature 422: 198-207
High-resolution, accurate, fast scanning MS: FT-MS
Hybrid linear ion trap FT-MS instruments
Electrostatic field:
Electromagnetic field:
LTQ-FTICR MS
Olsen JV et al., MCP2005
Non-destructive
Detection:
Olsen JV et al., MCP2004
k

m/ z
qB
v
1.535611  10 7  B
fc 


2r 2  m
m/z
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
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
960
344
202
26
11
# of tryptic
peptides for
m/z 1396.7813
Quantitation with Stable Isotope
Labeling
Unlabeled peptide:
Labeled peptide:
Element
Stable Isotope
1H
2H
12C
13C
14N
15N
16O
18O
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
Stable isotope dilution: same physico-chemical properties
Upregulated protein. Peptide ratio >1
”normal AA”
”heavy AA”
Arg-12C6
Arg-13C6
Resting cells
Treated (drug, GF)
Combine and lyse,
protein purification
or fractionation
Arg13C
6
Arg12C
6
Background protein. Peptide ratio 1:1
Arg12C
6
Arg13C
6
Proteolysis
(trypsin, Lys-C, etc.)
Quantitation and identification by MS
(nanoscale LC-MS/MS)
Ong SE et. al., Mol Cell Proteomics 2002
m/z
SILAC requirements
• 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:
NH2
C
H2N
H2
C
H
N
H2N
C
H2
CH
C
H2
NH
H2N
O
OH
C
O
L-arginine
Arg-13C6 (Δm=6 Da)
Arg-13C615N4 (Δm=10 Da)
H2C
CH2
H2C
CH
CH2
C
OH
L-lysine
Lys-13C6 (Δm=6 Da)
Lys-13C615N2 (Δm=8 Da)
• Growth supplements (e.g. dialyzed serum) if necessary
Quantitation Software –
http://msquant.sourceforge.net
Protein Quantitation
(Myosin IX)
No.
1
2
3
4
5
6
7
8
9
10
11
12
Peptide Sequence
GILTPR
WLVLR
NCAAYLR
NTNPNFVR
GALALEEKR
GDLPFVVTR
ALELDSNLYR
AGVLAHLEEER
LDPHLVLDQLR
VSHLLGINVTDFTR
AGKLDPHLVLDQLR
KQELEEICHDLEAR
Average Peptide Ratio
1.065±4.92%*
1.048±5.58%
0.999±4.54%
1.068±4.36%
1.055±5.80%
1.084±6.47%
1.024±4.91%
1.073±2.33%
0.992±2.61%
0.954±5.51%
1.073±2.68%
1.040±4.44%
Average Protein Abundance Ratio
SD
1.04
RSD (%)
3.82%
* Relative standard deviation
0.04
Gel-free phosphoproteome analysis workflow
Cell culture
days
Cell harvest
& trypsin
digestion
½ - 1 day
Strong cation
TiO2
exchange
Chromatography
Chromatography
pH<3 (bind)
pH<3
pH>10 (elute)
O.N.
½ day
½ day
LC-MS
pH~1
1-2 days
Data
Analysis
1-2 days
Phosphopetide enrichment by Titansphere (TiO2) chromatography
Competitive binding of peptides with DHB
<
<
Larsen et al. (2005) Mol Cell Proteomics 4:873-886
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
Hybrid linear ion trap FTICR MS:
LTQ FT (Thermo Scientific)
qB
v
1.535611  10 7  B
fc 


2r 2  m
m/z
LTQ-FT data-dependent experiments
Two Mass Spectrometers in one - High duty-cycle
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
LTQ-FT MS/MS optimized scan cycle:
FT-MS
MS-Full
IT-MS
SIM-MS 1st
MS2
0
300
SIM-MS 2nd
MS2
600
900
Time [msec]
1200
SIM-MS 3rd
Scan type
AGC
MS2
FT-MS Full
5,000,000
FT-MS SIM
50,000
IT-MS/MS
10,000
1500
1800
Phosphopeptide-directed MS3
Beausoleil SA et al. (2004) PNAS 101:12130-35.
Recent advances in FT-MS: LTQ-Orbitrap (Thermo)
Non-destructive
Detection:

FT:
LTQ:
Full
SIM1 SIM2 SIM3
MS2 MS3 MS2 MS3 MS2 MS3
0
1
Time [s]
2
Orbitrap:
LTQ:
Full scan
MS2 MS2 MS2 MS2 MS2
0
1
Time [s]
2
k
m/ z
LTQ-Orbitrap in the analysis of PTMs
„Hot“ CID
Multi-stage activation
CID with Multi-Stage Activation (MSA)
Pseudo
m/z
MS3
30ms
30ms
30ms
30ms
- 32.6 Da
- 49 Da
- 98 Da
wb
Precursor
Easy to identify multiply-phosphorylated peptides:
4, 5 and 6 phosphates
TiO2-enrichment of flow through from SCX (HeLa_EGF_CE_0_5_10)
CID in the C-trap (”Hot” CID or HCD)
Informative low mass ions – reporter ions
(Phosphotyrosine immonium ion, m/z = 216.0426)
Intracellular signaling networks (EGFR, HeLa)
(www.phosida.com)
• 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
Olsen et al. (2006) Cell 127(3):635-648
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
Ser/Thr/Tyr phosphorylation in B. subtilis
Bacillus
subtilis 168*
Previous studies
# of
genes
Expressed
4100
60%
(log)
P-proteins
P-sites
13
16
*Macek et al. 2006. Mol Cell Proteomics 6(4): 697-707
Ser/Thr/Tyr phosphorylation in B. subtilis
Bacillus
subtilis 168*
Previous studies
# of
genes
Expressed
4100
60%
(log)
This study
P-proteins
P-sites
P-proteins
P-sites
13
16
78
78
*Macek et al. 2006. Mol Cell Proteomics 6(4): 697-707
y*19 y*18 y*17
y14 y13
y11
y7 y6 y5 y4 y3 y2
Hpr protein V T A D pS G I H A R P A T V L V Q T A S K
y14++
100
90
80
740.427
Orbitrap full scan
C-trap MS/MS (HCD)
Precursor m=0.91ppm
Fragment m<2ppm
+++
y*18+++ y*19
70
Relative Abundance
y*18++
917.504
635.683
y*17+++
60
y13++
y7
573.662
50
40
y16++
y2
825.480
y4
234.145
406.229
30
y11
y14+++
y5
y*17
y3
20
1114.647
++
305.182
946.560
10
1017.598
0
200
300
400
500
600
700
m/z
800
900
1000
1100
CodY – Global regulator of transcription
y8 y7 y6 y5
90
80
Relative Abundance
70
60
50
pS V I V N A L R K
491.36
100
FT-ICR full scan
ion-trap MS/MS (CID)
Precursor m=6.39 ppm
Fragment m<0.5 Da
[M+2H]2+
-H3PO4
y3
b2 b3 b4
540.2988
b6
b8
40
30
20
0
505.00
283.00
10
350.55
186.82 213.09 254.27
200
300
400
700.27
566.18 601.27 643.27
500
600
m/z
MS3
b3
100
482.73
407.27
740.18 771.73 836.36 873.55
700
800
y5
936.73 982.09 1022.18 1062.18
900
1000
y6
601.31
700.30
282.13
90
80
70
Relative Abundance
y7++
60
y6++
50
254.20
40
407.44
b4
30
20
10
y3
y8
++
572.31
566.43
416.39 456.61
b2
185.10
213.15
169.10
381.19
301.41
461.97
683.42
613.40
835.35
712.48
667.45
499.42
813.50
854.58
877.74
769.45
0
150
200
250
300
350
400
450
500
b8
y7
b6
550
m/z
600
650
700
750
800
850
900
950
Phosphorylation in the main pathways
of carbohydrate metabolism (B. subtilis)
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)
Is S/T/Y phosphorylation common in bacteria?
Overview of prokaryotes studied so far
Previous studies
# of
genes
Expressed
Bacillus subtilis
168*
4100
Escherichia
coli K12**
This study
P-proteins
P-sites
P-proteins
P-sites
60%
(log)
13
16
78
78
4289
87%
(log)
20
12
79
81
Lactococcus
lactis
2250
?
(log)
1
1
52
68
Halobacterium
salinarum
2605
~80%
(stat)
1
1
18
15
E. coli vs. B. Subtilis phosphoproteome
• phosphoproteomes similar in:
• size
• distribution of S/T/Y phosphorylation
• classes of phosphorylated proteins
• increased essentiality
Essential
Essential
pT pY
phosphogenes
(%) (%)
proteins
(%)
(%)
67.9 23.5 8.6 17
>27
69.2 20.5 10.3 6.6
15.4
Genome No. of
No. of detected
pS
size
phospho- phosphorylation
(%)
(ORFs) proteins events
E. coli
B. subtilis
~4300
~4100
79
78
105
103
*Macek et al. 2007. submitted
Evolutionary conservation of bacterial
S/T/Y phosphoproteins
• 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
E. coli phosphoproteome
phosphoproteome
B. subtilis phosphoproteome
phosphoproteome
proteome
proteome
60
70
60
50
50
40
%
%
40
30
30
20
20
10
10
0
0
bacteria
eukaryotes
archaea
bacteria
eukaryotes
archaea
Evolutionary conservation of bacterial
S/T/Y phosphorylation sites
→ phosphoserine:
Conservation of phosphoserines - B. subtilis
Conservation of phosphoserine - E. coli
50
60.00
45
50.00
40
35
40.00
30.00
non-pS
%
%
30
pS
pS
25
non-pS
20
20.00
15
10
10.00
5
0.00
0
Bacteria
Eukaryotes
Archaea
Bacteria
Eukaryotes
Archaea
Evolutionary conservation of bacterial
S/T/Y phosphorylation sites
→ phosphothreonine:
Conservation of phosphothreonine - B. subtilis
70
60
50
40
%
pT
non-pT
30
20
10
0
Bacteria
Eukaryotes
Archaea
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?
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC):
Bacillus subtilis (Arg-, Lys-)
”normal AA”
”heavy AA” (+8Da)
Lys-13C615N2
Lys12C 14N
6
2
Treated cells
Peptide ratio 1:1 - No change.
Control cells
(succinate or low P)
Combine and lyse
GeLC-MS
Proteolysis
(trypsin)
m/z
Peptide ratio >1 - Downregulation.
Strong cation exchange
chromatography(SCX)
control
Titanium oxide chromatography
treated
nanoLC-MS/MS
(Quantitation and identification by MS)
log(2)
Dynamics of protein expression in B. subtilis :
Growth on succinate
10
9
8
7
6
5
4
3
2
1
0
-1 0
-2
-3
-4
-5
-6
-7
-8
-9
-10
argininosuccinate synthase
methionyl-tRNA synthetase
transcriptional regulator CodY
DNA polymerase III
succinyl-CoA synthetase
100
200
300
transcriptional regulator GutR
similar to phosphoglucomutase
400
500
600
glucose kinase
PTS glucose-specific enzyme II
beta-glucosidase
similar to phosphomannomutase
6-phospho-beta-glucosidase
700
Dynamics of protein phosphorylation in B. subtilis :
Growth on succinate
4
3
log(2)
2
protein
1
phospho
0
-1
-2
ybbT rsbW yerA ispU yvcT rocA fbaA rocD
protein
phospho
ptsH ptsH
(S12) (S46)
0.321 0.538 3.489 2.466 -0.19 0.072 0.469 0.246 0.225 0.225
0.6
0.407 2.593 1.059 0.804 -0.15 -0.43 -0.59 -0.32 -1.22
Growth on low succinate: Hpr protein
Ser46: pSIMGVMSLGIAK
GAEITISASGADENDALNALEETMK
Ser12: VTADpSGIHARPATVLVQTASK
GAEITISASGADENDALNALEETMK
NH2
COOH
S12 H15
S46
log(2)
Dynamics of protein expression in B. subtilis :
Growth under low PO4310
9
8
7
6
5
4
3
2
1
0
-1
-2 0
-3
-4
-5
-6
-7
-8
-9
-10
PTS sucrose-specific enzyme II
alkaline phosphatase A
inositol-monophosphate dehydrogenase
carbon starvation-induced protein
ATP synthase
GroEL
100
200
300
400
500
600
PTS enzyme I
DNA polymerase III
cysteine synthase
700
800
900
Dynamics of protein phosphorylation in B. subtilis :
Growth under low PO43-
4.00
2.00
log(2)
0.00
Protein
-2.00
Phospho
-4.00
-6.00
-8.00
ybbT ypfD sodA
Protein
ptsH ptsH
yfkK ypsB yvaB
(S46) (S12)
tpi
yvcT fbaA
pta
rocA
-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
Growth on low PO43-: Hpr protein
Ser46: pSIMGVMSLGIAK
YDADVNLEYNGK
Ser12: VTADpSGIHARPATVLVQTASK
YDADVNLEYNGK
NH2
COOH
S12 H15
S46
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?
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