Mass spectrometric methods to track protein phosphorylation and to characterize histone H2B variants

Abstract

Post-translational modifications (PTMs) act by changing a protein's structure or its interactions, and are important for expanding the repertoire of functions carried out by cellular proteins. Mass spectrometry (MS) is an important tool for analyzing PTMs because it can be used to identify and quantify proteins and their modifications without any prior knowledge of the sample content. This thesis describes two MS-based investigations of proteins and their PTMs. First, we optimized an approach for the Stable Isotope Labeling of Amino acids by Phosphate (SILAP) using [gamma-18O4]ATP to determine global site-specific phosphorylation rates in nucleo. We calculated approximate phosphorylation rate constants ranging from 0.34 min-1 to 0.001 min-1 based on labeling progress curves for over 500 protein phosphorylation sites. We then applied SILAP to determine sites that have different phosphorylation kinetics during G1/S and M phase of the cell cycle, and found that a small subset of functional phosphorylation sites involved in DNA replication and transcription were more actively phosphorylated at the G1/S phase boundary, whereas, sites involved in mitotic spindle phosphorylation were more actively phosphorylated during M phase. Second, we developed MS techniques to identify all somatic isoforms of histone H2B and to quantify nine of these isoforms in human cell lines. Variants and PTMs of histones H3 and H2A have important cellular functions and have been studied in depth; however, it remains unclear whether different H2B isoforms also have unique functions. Using the MS strategies that we developed, we identified differences in H2B isoform levels between cancer cell lines, suggesting cancer or tissue-specific H2B isoform regulation. We also found that specific, polyadenylated, H2B isoforms are expressed independently of the cell cycle. Finally, we immunoprecipitated epitope-tagged H2B1D, H2B3B, and H2B1C isoforms and found no H2B-isoform specific interactions.