Proteomics methods for probing molecular mechanisms in signal transduction.
mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes. Assessing the dynamic nature of this proteome is the challenge of modern proteomics. Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions. Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins. These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation. High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches. In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches. Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction.
Department of Dairy Science, University of Wisconsin, Madison 53706, USA. lgsheffi@facstaff.wisc.edu
L G
LG
Sheffield
J J
JJ
Gavinski
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D010449:Peptide Mapping; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome; D012333:RNA, Messenger