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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

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)
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)
Kindlin Ectopic expression in HEK cells 4 phospho-sites in 79.4% coverage of 645 aa
Data available		 Parsons (Fox)
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 - Webb et al., 2005 (CMC Update Article); Schroeder et al., 2005; 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		 Parsons, Hoffman (Hunt, Beckerle)
Gelsolin Ectopic expression in HEK cells In progress Parsons (Fox)
LIMK1 Ectopic expression in HEK cells In progress Parsons (Fox)
Myosin II Ectopic expression in HEK cells In progress Horwitz (Hunt)
PINCH Ectopic expression in HEK cells In progress Parsons (Fox)
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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
Data available - 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; Udeshi et al., 2007, software development in progress; Chi et al., 2007 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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