Optimization of mass spectrometry-compatible surfactants for shotgun proteomics.
An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.
Chen
Emily I
EI
Department of Cell Biology, 10550 North Torrey Pines Road, SR11, The Scripps Research Institute, La Jolla, California 92037, USA.
Cociorva
Daniel
D
Norris
Jeremy L
JL
Yates
John R
JR
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D010179:Pancreas / Q000737:chemistry / Q000166:cytology; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D012996:Solutions; D012997:Solvents / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
D002457:Cell Extracts; D010455:Peptides; D012996:Solutions; D012997:Solvents; D013501:Surface-Active Agents; D014357:Trypsin