Lysine post-translational modification analysis and quantitation using chemically derivatized analogs

The goal of this work was to develop qualitative and quantitative mass spectrometric tools for the elucidation of the histone code. The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation.;In the first phase of this work, levels of ubiquitination were quantitated using a structurally homologous label that is chemically similar to the GlyGly tag, which is left at the ubiquitination site upon trypsinolysis. A glycinylation protocol was established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. The technique was then used to quantitate the increase in mono-ubiquitinated histone H2B that is observed in yeast which lack the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast.;A biotinyl-lysine derivatization method was previously developed in the lab for the identification of new substrates of the sirtuin family of lysine deacetylases. A time course experiment showed that Sir2 preferentially deacetylated H3-K79 compared to Hst2. This observation led to the second phase of this work that involved the search for in vivo acetylated H3-K79, which had not yet been observed in yeast. Exact mass measurements were used to correctly assign the acetylation modification observed in a DeltaDot1DeltaSir2 strain. We also attempted to identify the histone acetyltransferase (HAT) that is responsible for acetylating the H3-K79 position by knocking out different HATs in the DeltaDot1DeltaSir2 strain.;In the final phase of this work, the deuteroacetylation technique was used in combination with retention times and immonium ions for the differentiation of trimethylation and acetylation without the need for high mass resolution or mass accuracy. It was also demonstrated how the deuteroacetylation technique can be used for the quantitation of acetylation levels even when another modification is present on the same peptide. This combinatorial aspect of quantitation could prove extremely useful when attempting to decipher the histone code.