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Proteomics - Activities

Objectives

  • to develop proteomic methodologies for identifying migration related proteins, their binding partners, phosphorylation sites and post-translational modifications
  • to determine the effect of growth factor and adhesion on the binding partners, utilized phosphorylation sites and post-translational modifications for a subset of migration proteins

Proteomics Protocols

Phosphoproteomics | Binding Partners | Organelles | MS Technology Developments

Phosphoproteomics

Target Protein Method Status / Publication Contact (Collaborators)
bPix Ectopic expression in HEK cells 10 phospho-sites in 88.7% coverage of 675 aa
Data available - Mayhew et al., 2007 (CMC Update Article)		 Horwitz (Hunt)
Calpain 2 Ectopic expression in HEK cells 4 phospho-sites in 88.1% coverage of 708 aa
Data available		 Parsons (Fox)
CCM2 20 sites in 98.2% coverage of 444 amino acids
Data available		 Ginsberg (Fox)
Cortactin Ectopic expression in HEK cells 18 phospho-sites in 89.7% coverage of 543 aa
Data available - Martin et al., 2006		 Parsons (Hunt)
Dynamin 2 Ectopic expression in HEK cells 8 phospho-sites in 89.4% coverage of 879 aa
Data available		 Parsons (Fox)
FAK Ectopic expression in HEK cells 18 phospho-sites in 80.2% coverage of 1045 aa
Data available - Grigera et al., 2005 (CMC Update Article)		 Parsons (Hunt)
Filamin-a Endogenous from human platelet 11 phospho-sites in 82.5% coverage of 2630 aa
Data available		 Ginsberg (Hunt)
GIT1 Ectopic expression in HEK cells 28 phospho-sites in 89.9% coverage of 773 aa
Data available - Webb et al., 2006 (CMC Update Article); Webb et al., 2006 (CMC Update Article)		 Horwitz (Hunt)
ICAP1 7 sites in 99.5% coverage of 194 amino acids
Data available		 Ginsberg (Fox)
Kindlin Ectopic expression in HEK cells 4 phospho-sites in 79.4% coverage of 645 aa
Data available		 Parsons (Fox)
KRIT1 2 sites in 99.5% coverage of 735 amino acids
Data available		 Ginsberg (Fox)
MYH10 - (MIIB) Ectopic expression in HEK cells 16 sites in 79.0% coverage of 1972 amino acids
Data available		 Horwitz (Hunt)
MYH9 - (MIIA) Ectopic expression in HEK cells 9 sites in 75.1% coverage of 1952 amino acids
Data available		 Horwitz (Hunt)
Myosin light chain/MYL9 Ectopic expression in HEK cells 4 phospho-sites in 98.3% coverage of 172 aa
Data available		 Horwitz (Hunt)
N-Wasp Ectopic expression in HEK cells 6 phospho-sites in 87.7% coverage of 505 aa
Data available		 Parsons (Fox)
p115 Rho GEF Ectopic expression in HEK cells 15 phospho-sites in 91.7% coverage of 867 aa
Data available		 Parsons (Fox)
p130Cas Myc-tagged and expressed in HEK cells 9 phospho-sites in 87.0% coverage of 977 aa
Data available		 Parsons (Fox)
Pak1 Ectopic expression in HEK cells 30 phospho-sites in 80.5% coverage of 529 aa
Data available - Mayhew et al., 2007 (CMC Update Article)		 Horwitz
Parvin Ectopic expression in HEK cells 4 phospho-sites in 88.7% coverage of 380 aa
Data available		 Parsons (Fox)
Paxillin Ectopic expression in HEK cells 38 phospho-sites in 79.4% coverage of 791 aa
Data available - Schroeder et al., 2005; Webb et al., 2005 (CMC Update Article); Nayal et al., 2006 (CMC Update Article)		 Horwitz (Hunt)
Talin Endogenous from human platelet 30 phospho-sites in 95.1% coverage of 2540 aa
Data available - Ratnikov et al., 2005 (CMC Update Article)		 Ginsberg (Hunt, Liddington)
VAV1 Ectopic expression in HEK cells 1 phospho-site in 86.1% coverage of 858 aa
Data available		 Parsons (Fox)
Vinculin Ectopic expression in HEK cells 2 phospho-sites in 92.1% coverage of 1062 aa
Data available		 Parsons (Fox)
Wasp Ectopic expression in HEK cells 8 phospho-sites in 85.8% coverage of 515 aa
Data available		 Parsons (Fox)
Zyxin TAP-tagged GST-Zyxin 4 phospho-sites in 78.0% coverage of 564 aa
Data available		 Hoffman, Parsons (Hunt, Beckerle)
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Binding Partners

Target Protein Method Status / Publication Contact (Collaborators)
beta subunit of CaMK-II / delta subunit of CaMK-II Ectopic expression in NIH 3T3 cells 9 CaMK-II binding proteins identified by ectopic expression of FLAG-tagged CaMK-II splice variants
Data available - Seward et al., 2008		 Horwitz
Calpain 2 Ectopic expression in HEK cells Proteins co-purifying with expressed FLAG-tagged calpain
Data available		 Horwitz
GIT1 Ectopic expression in HEK cells 7 proteins exclusively co-purified with Git1
Completed - Mayhew et al., 2006 (CMC Update Article); Webb et al., 2006 (CMC Update Article)		 Horwitz
H-Ras 12V Ectopic expression in NIH 3T3 cells 247 co-purified proteins validated
Data available - Goldfinger et al., 2007 (CMC Update Article)		 Ginsberg (Hunt)
Pak1 Ectopic expression in HEK cells 13 proteins exclusively co-purified with Pak1
Data available - Mayhew et al., 2006 (CMC Update Article)		 Horwitz
Paxillin Ectopic expression in HEK cells 10 co-purifying proteins validated
Data available - Schroeder et al., 2005; Mayhew et al., 2006 (CMC Update Article)		 Horwitz
Pix Ectopic expression in HEK cells 7 proteins exclusively co-purified with Pix
Data available - Mayhew et al., 2006 (CMC Update Article)		 Horwitz (Hunt)
R-Ras38V Ectopic expression in NIH 3T3 cells 25 co-purified proteins validated
Data available - Goldfinger et al., 2006 (CMC Update Article); Goldfinger et al., 2007 (CMC Update Article)		 Ginsberg (Hunt)
Rac1 Ectopic expression in HEK cells Data available Horwitz
Rap1A(G12V) Ectopic expression in NIH 3T3 cells 50 co-purified proteins validated
Data available - Goldfinger et al., 2007 (CMC Update Article)		 Ginsberg (Hunt)
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Organelles

Target Organelle Method Status / Publication Contact (Collaborators)
Lamellipodium Purification & characterization of psuedopodium phosphoproteome 1,073 Pseudopodium-enriched and 1,088 Cell Body -enriched proteins found
Data available - Wang et al., 2007 (CMC Update Article); Wang et al., 2007 (CMC Update Article); Wang and Klemke, 2008 (CMC Update Article)		 Klemke
Stress Fibers stress fiber isolation protocol 207 proteins identified in stress fibers
Data available - download stress fiber analysis		 Burridge
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Mass Spectrometry Technology Developments

MS Technology Status / Publication Contact (Collaborators)
Differential analysis of phosphopeptide expression Completed - Schroeder et al., 2005 Hunt
ETD fragmentation improvements Completed - ETD fragmentation - instrumentation complete Syka et al., 2004; Coon et al., 2005; Coon et al., 2005; Chi et al., 2007; Udeshi et al., 2007, software development in progress Hunt
IMAC with methyl ester treatment (enhanced selection of phosphopeptides) Completed - Garcia et al., 2005 Hunt
LTQ-FTMS improvements Completed - QLT-FTMS - instrumentation complete Syka et al., 2004 Hunt
Methods for full protein sequence mapping Completed Hunt
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