Characterization of protein phosphorylation by nanoflow HPLC-microelectrospray ionization mass spectrometry

The regulation of protein phosphorylation is critical for proper cellular function, but conventional methods for the study of this posttranslational modification are limited in their ability to provide direct, routine, and global detection of phosphorylation sites. Mass spectrometry is an analytical tool that is well suited for the direct identification of phosphorylation because it can be combined with phosphopeptide enrichment strategies such as immobilized metal affinity chromatography (IMAC) to detect the complement of phosphorylation sites of a single protein or complex mixtures of proteins. Additionally, peak areas from chromatographically-separated peptides and/or phosphopeptides detected by the mass spectrometer can be used to measure changes in phosphorylation abundances after cellular perturbation.; Chapter 1 provides an introduction to protein phosphorylation and mass spectrometry. The remaining chapters describe methods for the routine detection and characterization of protein phosphorylation using mass spectrometry. Specifically, the second chapter describes the study of tristetraprolin (TTP), an RNA-binding phosphoprotein involved in regulating the inflammatory response, using a 3D-ion trap mass spectrometer. Comparison of phosphorylated to nonphosphorylated peptide chromatographic peak areas from cells transfected with wild type or kinase defective MAP Kap Kinase 2 (MK2) allowed us to determine that TTP in an in vivo substrate for MK2. The third chapter describes the study of paxillin and GIT1, two focal adhesion proteins involved in cell migration, using a hybrid 2D linear ion trap with Fourier transform mass spectrometry. Here, direct comparison of phosphopeptides abundances was achieved via differential isotopic labeling of phosphopeptides derived from differentially-treated cells and enrichment by IMAC. Finally, the fourth chapter describes the application of two new peptide/phosphopeptide fragmentation techniques, Pseudo MSn and electron transfer dissociation (ETD), which greatly enhance the quality of data obtained from the analysis of phosphopeptides by mass spectrometry.