P77-M Automated Analysis of Gel-Derived Phosphoproteins Using the Investigator Proteomic System

摘要

The evolution of proteomics-based technologies has led to the development of powerful analytical methods for the analysis of post-translational modifications such as phosphorylation. Chemical derivatization strategies that involve beta-elimination and Michael addition chemistries as well as chromatographic enrichment approaches using immobilized metal affinity chromatography have been successfully applied to global phosphoproteome analysis. Recently, enrichment of phosphorylated peptides using TiO2 prior to mass spectrometric analysis has been shown to be a selective and robust method for phosphoprotein characterization. We present an automated workflow using TiO2 microcolumns for the enrichment of phosphorylated peptides derived from in-gel digested proteins in combination with the Investigator Proteomic System (Genomics Solutions, Ann Arbor, MI).A pool of beta casein and ovalbumin (10 μg each) was used as a standard to test the automated phosphoprotein analysis workflow. This same workflow was then successfully applied to characterize in vitro and in vivo phosphorylation events of selected 14-3-3 proteins derived from Arabidopsis thaliana. Samples were separated by either 1D or 2D SDS-PAGE, stained with Pro-Q Diamond (phosphoprotein-specific) fluorescent stain, and imaged using either a Typhoon 9400 scanner or the Investigator ProPic (Genomic Solutions). Putative phosphoprotein-containing spots were then excised and digested in-gel with trypsin using the Investigator ProPic and ProGest, respectively. Tryptic digests from each protein spot were processed with the ProMS workstation using TiO2 microcolumns (Glygen Corp., Columbia, MD) for phosphopeptide enrichment prior to MALDI-TOF/TOF analysis (ABI 4700 Proteomics Analyzer).