Identification of p65-associated phosphoproteins by mass spectrometry after on-plate phosphopeptide enrichment using polymer-oxotitanium films

Abstract

Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulusspecific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spe..