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HUPO Conference, September 5-10, 2007, Seoul, Korea.

Analysis of post-translational modifications by mass spectrometry

Ole N. Jensen, Ph.D.

Lundbeck Foundation Professor Protein Research Group Dept. Biochemistry & Molecular Biology University of Southern Denmark [email protected] www.protein.sdu.dk

Genome size of model organisms

Organism Yeast: Saccharomyces cerevisiae Fruit fly: Drosophila melanogaster Nematode: Caenorhaptidis elegans Thale cress: Arabidopsis thaliana Human: Homo sapiens No. genes 6.100 13.000 17.000 19.000 23.000

Functional Genomics and Proteomics

Gene exon1 exon2 exon3

Transcription

exon4

Transcript

exon1

exon2

exon3

Alternative splicing

exon4

mRNA

exon1

exon2

exon3

exon1

Translation

exon2

exon4

Gene products

PTM: Post Translational Modifications

Glycosylation Lipid anchor Ubiquitination

P

Phosphorylation

Functional genomics and proteomics: From genes to gene products

DNA mRNA Protein

Co/post-translational processing Alternative splicing

Gene

mRNA1 mRNA2 mRNA3 mRNA4 mRNA5

Protein1 Protein2 Protein3 Protein4 Protein5 Protein6 .... Proteini

Jensen (2004) Curr. Opin. Chem. Biol.

Post-translational modifications

Modification-specific proteomics:

Interpreting the biological roles of PTMs

Identify PTM'ed proteins Determine PTM sites of proteins Determine functional PTMs by site-specific quantitation Determine interdependence and cooperativity between multiple PTMs Model/simulate complex molecular systems

Mass spectrometry

· A mass spectrometer generates gas-phase ions from a sample, separates them according to their mass-to-charge ratio (m/z) and generates a record of their abundance

­ ­ ­ ­ Molecular weight determination of peptides and proteins Amino acid sequencing of peptides Detection of post-translational modifications (m) Relative quantitation of peptides and proteins

Tandem mass spectrometry (MS/MS)

Ion Source Mass Analysis

Collision induced Dissociation

Mass Analysis

1

2

Intensity

Ion Detector

m/z

Mass Spectra

(MS and MSMS data)

Computer

Peptide sequencing by MS/MS Peptide analysis by MS

1 1 N

PT M

1

C

M 2 M+m

2 N

C m PTM

2

PT M

- Amino acid residue modified with PTM - Amino acid residue C - C-terminal amino acid residue N - N-terminal amino acid residue

m, PTM amino acid assignment

Larsen et al (2006) Biotechniques 40 (6) 790-798

PTM-specific ion signal

C

MS/MS

N Stable PTM m( PTM, position )

PT M

PTM-specific neutral loss

PTM

MS/MS/MS

C

MS/MS

N Labile PTM m(PTM =

PT M

PTM

)

(aa)

C - C-terminal amino acid residue N - N-terminal amino acid residue

PTM

- Amino acid residue modified with stable PTM - Amino acid residue modified with labile PTM

LC-MS/MS analysis of En-2

y6 y5 y4

y2 y1

265- R Q Sp L A Q E L S L N E S Q I K-280

En-2 10a U01276AS 339

b2 b3

b5

b7 b8

914.1

b10 b11 b12

100

[M+2H-H3PO4] 2+

%

y2

216.1

260.2 285.2

354.2

467.3 475.3

441.2

538.4

604.4 636.4 676.9 718.5

795.5

1006.6

y1

147.1 0

1108.7

*

100 200 300 400 500

600

700

800

900

m/z 1000 1100 1200 1300 1400

1222.8

1351.8

327.2

y5

y6 b* 7

963.1

b*3 b2

y4 b5

*

[M+2H]2+

* b* b11 10

b* 12

MR Larsen et al (2006) Biotechniques

Protein and proteome analysis by mass spectrometry

Sample preparation

Voyager Spec #1[BP = 1203.6, 16210]

100 90 80 70 1204.5889 % Intensity 60 50 40 30 1205.5986 20 10 0 1203.08660 1203.5884 1.6E+4

1203.78144

1204.47628 Mass (m/z)

1205.17112

1205.86596

1206.56080

Sample (Proteins, peptides)

Ionisation and mass analysis (MALDI, ESI)

Mass spectrum

Biological material - Tissue - Cells - Recombinant protein

Protein structure information

Protein function

Modification-specific proteomics

Optimized sample preparation for PTM-peptides

MS data acquisition

MS (m) MS/MS sequencing MS/MS neutral loss MS/MS diagnostic ions Multistage MS

MALDI MS/MS ESI MS/MS

Mass spectra Organelle/complex purification, and/or PTM protein enrichment, and/or PTM peptide enrichment

Computational data analysis and data mining

PTM assignments

PTM function

Validation

Sample preparation for MS Desalting - Enrichment - Concentration

10 l micropipettor tip Syringe (1.25 ml) · · · · · POROS R1, R2, or OligoR3 NTA-silica [Fe(III)-IMAC] HILIC Graphite TiO2

1 mm

C4/C18

Millipore ZipTip

GELoader tip

Gobom et al (1999) J Mass Spectrom. 34 105-16. Stensballe et al (2001) Proteomics Larsen et al (2002) Proteomics; Larsen et al (2005, 2007) Mol. Cell. Proteomics Hägglund et al (2004, 2007) J. Proteome Res. , Omaetxebarria et al (2006) Anal. Chem. Rappsilber et al (2003) Anal. Chem.

Nano-column

StageTip

Phosphorylation of Pil1 promotes eisosome formation

MALDI peptide mass mapping of Pil1 (S. cerevisiae) 60%

CIP

-

+

59%

Combined a.a. sequence coverage: 91% Max. achievable a.a. sequence coverage: 93%

44%

Analysis of Pil1 by mass spectrometry revealed 14 phosphopeptides (7+1 phosphorylation sites)

LC-ESI-MS/MS (LTQ-FT-ICR MS, NL-MS3) Fe(III)-IMAC + MALDI-QTOF-MS/MS MALDI MS + nanoESI-QTOF-MS/MS

Trypsin Asp-N

(Endo LysC sample produced no phosphopeptide data!)

(Gruhler et al, submitted)

ECD/ETD MS/MS of phosphopeptides

Agilent HCT Ultra/ETD iontrap

Phosphoproteome analysis by ETD/CID MS/MS

· ETD MS/MS (3+, 4+, ..)

­ Generate larger peptides

· Endoproteinase Lys-C · Limited proteolysis by trypsin

(also, many missed cleavages due to phosphorylation)

­ Extensive fragmentation (c, z´) ­ ETD seems more efficient than CID for phosphopeptide sequencing by ion trap MS ­ CID and ETD generate distinct datasets

Chi et al (2007) PNAS 104 (7) 2193-2198 Molina et al (2007) PNAS 104 (7) 2199-2204 Coon et al, ASMS 2007

Stability of PTMs: The case of phosphohistidine

Anal. Chem. (2007) 79 (19) 7450-7456

MS/MS sequencing of phosphohistidine peptides

ETD/ECD enable sequencing of pHis peptides

Kleinnijenhuis et al (2007) Anal. Chem. 79 (19) 7450-7456

Membrane proteomics and post-translational modifications

PTM analysis by Shave-and-Conquer strategy ­ ­ GPI-anchored membrane proteins (A.th, HeLa)

Elortza et al (2003) Mol. Cell. Proteomics Elortza et al (2006) J. Proteome Res.

Plasma membrane phosphoproteomics (A.th.)

Nühse et al (2003) Mol. Cell. Proteomics Nühse et al (2004) Plant Cell

Quantitative proteomics of membrane proteins during stem cell differentiation

Foster et al (2005) Stem Cell

Quantitative phosphoproteomics of the yeast pheromone response

Gruhler et al (2005) Mol. Cell. Proteomics

Differential, multisite phosphorylation of the EGF receptor

Boeri Erba et al (2006) Mol. Cell. Proteomics Boeri Erba et al (2007) J. Proteome Res.

Modification-specific proteomics

· SHAVE and CONQUER:

Determination of post-translationally modified membrane proteins

­ GPI-anchored membrane proteins

Elortza et al. (2003) Mol. Cell. Proteomics 2 (12) 1261-1270. Elortza et al (2006) J. Proteome Res.

­ Membrane phosphoproteomics

Nühse et al. (2003) Mol. Cell. Proteomics 2 (11) 1234-1243. Nühse et al. (2004) Plant Cell

www.protein.sdu.dk

Post-translational modifications

Chromatin dynamics

Me Ac Ac Ac 3Me Ac

* * *

| 8

*

*

*

* Ac -N-SGRGKGGKGLGKGGAKRHRKILRDNIQGITKPAIRR3 5 12 16 20

| |

|

|

|

MS/MS: Diagnostic ions (acetylated lysine: m/z 126.1)

Analysis of histone acetylation upon treatment w. HDAC inhibitor

In solution digest

9000 H4(4-17)K5acK8acK12acK16ac 8000

7000

6000

relative intensity

5000

4000

3000

2000

1000

0

-1000 PXD101 treatment

Beck et al (2006) Mol. Cell. Proteomics 5 (7) 1314-1325

Comparative LC-MS/MS analysis of histone acetylation upon treatment w. HDAC inhibitor

0 0.1 0.3 1 3 10 M

Beck et al (2006) Mol. Cell. Proteomics 5 (7) 1314-1325

TiO2 enrichment of PTM'ed peptides

MR Larsen et al, MCP, 2005, 2007

TiO2 enrichment of sialic acid containing glycopeptides

Depleted plasma +phosphatase no enrichment

Depleted plasma +phosphatase +TiO2 enrichment

MR Larsen et al, MCP, 2007

Modification-specific proteomics

Optimized sample preparation for PTM-peptides

MS data acquisition

MS (m) MS/MS sequencing MS/MS neutral loss MS/MS diagnostic ions Multistage MS

MALDI MS/MS ESI MS/MS

Mass spectra Organelle/complex purification, and/or PTM protein enrichment, and/or PTM peptide enrichment

Computational data analysis and data mining

PTM assignments

PTM function

Validation

Quantitative proteomics by mass spectrometry

Peptide intensity profiling

Stable isotope labeling (SIL)

Stable Isotope Labeling in Cell Culture (SILAC)

Ong et al., MCP, 1, 376-386 (2002) Zhu et al., RCM 16, 2115-23 (2002) arginine lysine

13C 13C 6-arginine 6-lysine

Intensity

t y i s e n I

Cell lysis

Proteolysis Protein Mixture MS

M/z

Site specific differential phosphorylation of EGF receptor upon stimulation

GSHQISLDNPDpYQQDFFPK RT = 81.2, z=+3, m/z=772.82/774.82 LLGAEEKEpYHAEGGK RT = 49.1, z=+3, m/z=570.99/575

774.82 772.82

Ratio: 1.2

Ratio: 3.0

575.00

570.99

772 773 774 775 776 777

570 571 572 573 574 575 576 577

SILAC and LC-ESI-MS/MS

Modification-specific proteomics

Optimized sample preparation for PTM-peptides

MS data acquisition

MS (m) MS/MS sequencing MS/MS neutral loss MS/MS diagnostic ions Multistage MS

MALDI MS/MS ESI MS/MS

Mass spectra Organelle/complex purification, and/or PTM protein enrichment, and/or PTM peptide enrichment

Computational data analysis and data mining

PTM assignments

PTM function

Validation

Conclusion

· Quantitation of proteins and of protein phosphorylation and other PTMs is feasible by using MALDI MS and ESI MS · Integrated, modification-specific workflows increases the analytical performance dramatically · In-depth quantitative proteomics is required to identify differentially regulated PTMs · Quantitative (phospho)proteomics is a new approach to study cell signaling pathways and understand complex biological phenomena

Cells/tissue Protein preparation (lysate/organelle/complex) Shotgun sequencing Digestion A Digestion B Digestion C Digestion PTM-based affinity enrichment MS/MS, MSn MS/MS, MSn Digestion PTM-peptide release MS/MS, MSn Modification-specific analysis PTM-based affinity enrichment PTM-based covalent capture

Sequence Database searching

PTM assignments

Take home messages

· MS is uniquely suited for characterization and quantitation of post-translational modifications · Modification-specific analytical strategies have proven successful, but

­ There are no 'easy' protocols or methods! ­ Careful optimization of sample preparation protocols and MS/MS setup is required ­ Data analysis is demanding

Acknowledgements

Protein Research Group University of Southern Denmark

Elisabetta Boeri-Erba Albrect Gruhler Shabaz Mohammed Allan Stensballe Ole Hørning Emmanuelle Sachon Nicolai Bache Tine E. Thingholm Søren S. Jensen Iris Stulemeijer Peter Højrup Thomas J.D. Jørgensen Finn Kirpekar Martin R. Larsen Peter Roepstorff

EGFr

Paola DeFilippi, Univ. Torino, IT Karin Hjernø Miren Josu Omatxebarria Felix Elortza Morten B. Trelle Jakob Bunkenborg Rune Matthiesen Christian Ravnsborg Søren Andersen Kate Rafn Lene Skou Inger Christiansen Andrea Lorentzen Helle M. Mortensen

Histones

Adriana Salcado, Henk Stunnenberg University of Nijmegen, NL TopoTarget A/S Technological institute

...many collaborators in Denmark and abroad

Instrument manufacturers Danish Biotechnology Instrument Center European Union Danish Research Councils Lundbeck Foundation

Protein Research Group University of Southern Denmark

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Proteomics

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