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

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)

Quantitation Software – http://msquant.sourceforge.net

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)

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

Larsen et al. (2005) Mol Cell Proteomics 4:873-886

Phosphopetide enrichment by Titansphere (TiO2) 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 C18 HPLC columns• flow ~250 nL/min

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

m/z

B101.535611

m2

Bq

2

7

r

vf c

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

Phosphopeptide-directed MS3

Beausoleil SA et al. (2004) PNAS 101:12130-35.

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

LTQ-Orbitrap in the analysis of PTMs

Multi-stage activation

„Hot“ CID

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

Informative low mass ions – reporter ions(Phosphotyrosine immonium ion, m/z = 216.0426)

CID in the C-trap (”Hot” CID or HCD)

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

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

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

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)

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

• 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-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

-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

-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

Ser46: pSIMGVMSLGIAK

Ser12: VTADpSGIHARPATVLVQTASK

Growth on low succinate: Hpr protein

COOHNH2

S12 H15 S46

GAEITISASGADENDALNALEETMK

GAEITISASGADENDALNALEETMK

-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-

-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-

Ser46: pSIMGVMSLGIAK

Ser12: VTADpSGIHARPATVLVQTASK

YDADVNLEYNGK

YDADVNLEYNGK

Growth on low PO43-: Hpr protein

COOHNH2

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 BiochemistryMatthias MannFlorian GnadJesper V. OlsenChanchal Kumar

Technical University of DenmarkIvan MijakovicBoumediene SoufiDina Petranovic

Thermo ScientificStevan HorningOliver Lange