phosphoproteomics and motif mining martin miller ph.d. student cbs dtu miller@cbs.dtu.dk

Phosphoproteomics and motif mining

Martin MillerPh.d. studentCBS DTUmiller@cbs.dtu.dk

Outline

MS-based phosphoproteomicssubstrate-motifs in intracellular signallingresearch project: motif decomposition of the phosphotyrosine proteome

Mass spectrometry-based proteomics

Aebersold & Mann, Nature 422: 198-207, 2004.Aebersold & Mann, Nature 422: 198-207, 2004.

Select one peptide species

Collide

Separate fragments

Y3

Y4

Y5

Y6

Y7

Mass spectrometry-based proteomics

PTM detection using MS

Quantitative phosphoproteomicsusing SILAC

Growth media lacking the SILAC labeling amino acid (e.g. Arg)

Stable Isotope Labeled Amino Acids:

Δm=6 Da Δm=10 Da

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

State A State B

Light Isotope Heavy Isotope

Mix 1:1

Optional Protein Fractionation

Digest with Trypsin

Protein Identification and Quantitation by LC-MS

Ong Ong et alet al., Mol. Cell. Proteomics 1, 2002.., Mol. Cell. Proteomics 1, 2002.

Typical SILAC experiment workflow

Upregulated protein - Peptide ratio >1

Background protein - Peptide ratio 1:1

Arg-12C6

Arg-13C6

m/z

Arg-12C6

Arg-13C6

m/z

m/z

Arg-12C6

Arg-13C6

m/z

Arg-12C6

Arg-13C6

SILAC labeling for quantitation

ConvenientNo extra step introduced to experiment, just slightly different growth medium

All identified proteins are - in principle – quantifyableQuantitation of proteins affected by different stimuli, disruption of genes, etc.Quantitation of post-translational modifications (phosphorylation, etc.)

Fishing for modification-dependent interactors using a bait sequence

Asp-Ser-Trp-Ala-Arg-Leu-His-Gly-Tyr-Met-Ile-Met-Glu-Pro-Lyssolidsupport

Asp-Ser-Trp-Ala-Arg-Leu-His-Gly-Tyr-Met-Ile-Met-Glu-Pro-Lyssolidsupport

P

phosphorylation specific pull-down experiments

Schulze W and Mann M. (2004)JBC 2004

Advantages of the SILAC pull-down method

No overexpression – no taggingStraightforward separation between specific interactors and background bindersDetection of low abundance and moderate affinity interactorsEspecially suited for PTM interaction studiesDetermination of the exact interaction site within the proteinImportant protein-protein interactions in cell signaling are frequently mediated by short, unstructured sequences – linear motifs

sequence motifs in intracellular signalling

linear peptides sequence motifs guide signalling• kinases• phosphorylation-

dependent interaction domains (SH2, PTB, 14-3-3 etc.)

directionality and specificity

Kinome tree and kinase substrates

Linding et al, Cell, accepted

SH2 domain tree

A specific branch of the SH2 domain tree

The Phospho.ELM database currently contains 13614 phosphorylation sites in 4421 eukaryotic proteins. However, only ~23% of have know function.

Thus there is a unique opportunity to mine for novel phosphorylation motifs

“The Widening Gap”

Research protect

motif decomposition of the phosphotyrosine proteome

A new method for clustering uncharacterized phosphopeptides and mining for novel phosphorylation motifs

following slides are erased because the data is confidential since results are not published yet