Phosphoproteomics and motif mining

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Phosphoproteomics and motif mining

• Martin Miller
• Ph.d. student
• CBS DTU
• miller_at_cbs.dtu.dk

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Outline

• MS-based phosphoproteomics
• substrate-motifs in intracellular signalling
• research project motif decomposition of the
  phosphotyrosine proteome

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Mass spectrometry-based proteomics
Select one peptide species
Collide
Separate fragments
Y7
Y6
Y5
Y4
Y3
Aebersold Mann, Nature 422 198-207, 2004.
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Mass spectrometry-based proteomics
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PTM detection using MS
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Quantitative phosphoproteomicsusing SILAC
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Stable Isotope Labeling with Amino Acids in Cell
Culture (SILAC)
Growth media lacking the SILAC labeling amino
acid (e.g. Arg) Stable Isotope Labeled Amino
Acids
?m6 Da
?m10 Da
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Stable Isotope Labeling with Amino Acids in Cell
Culture (SILAC)
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Typical SILAC experiment workflow
Upregulated protein - Peptide ratio gt1
Background protein - Peptide ratio 11
Ong et al., Mol. Cell. Proteomics 1, 2002.
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SILAC labeling for quantitation

• Convenient
• No extra step introduced to experiment, just
  slightly different growth medium
• All identified proteins are - in principle
  quantifyable
• Quantitation of proteins affected by different
  stimuli, disruption of genes, etc.
• Quantitation of post-translational modifications
  (phosphorylation, etc.)

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Fishing for modification-dependent interactors
using a bait sequence
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phosphorylation specific pull-down experiments
Schulze W and Mann M. (2004) JBC 2004
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Advantages of the SILAC pull-down method

• No overexpression no tagging
• Straightforward separation between specific
  interactors and background binders
• Detection of low abundance and moderate affinity
  interactors
• Especially suited for PTM interaction studies
• Determination of the exact interaction site
  within the protein
• Important protein-protein interactions in cell
  signaling are frequently mediated by short,
  unstructured sequences linear motifs

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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

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Kinome tree and kinase substrates
Linding et al, Cell, accepted
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SH2 domain tree
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A specific branch of the SH2 domain tree
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The Widening Gap
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
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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