phosphoproteomics and motif mining martin miller ph.d. student cbs dtu [email protected]
TRANSCRIPT
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