Mass spectrometric analysis of post-translational modifications of histone and histone-like proteins

Post-translational modifications (PTMs) play a critical role in many biological processes, such as gene expression, cell signaling and mitosis. Alterations to these processes are known to be characteristic of cellular disease states, most notably cancer. It is crucial to characterize the PTMs of proteins that mediate these cellular processes. Mass spectrometry is a powerful tool for studying PTMs. Presented herein are three examples of the imperative use of mass spectrometry to characterize the PTMs of histone and histone-like proteins to help solve biological problems. The high resolution mass spectrometer, LTQ-FTMS, offers accurate mass measurement. Tandem mass spectrometry using collision activated dissociation or electron transfer dissociation, a newly developed fragmentation technique, make it possible to sequence peptides and unambiguously assign the specific sites of modification.; The first study focused on the investigation of heterochromatin; the regions of chromatin that contain transcriptionally silenced genes. Methylation of histone H3, catalyzed by methyltransferases, and recruitment of chromodomain-containing proteins are linked to heterochromatin formation. To identify the lysine substrates of specific methyltransferases, modification patterns of H3 isolated from WT Tetrahymena were compared to the modification patterns of H3 isolated from a mutated Tetrahymena strain, in which a gene that encodes for a specific methyltransferase was knocked out.; In the second study, a global analysis was performed on the histone-like protein, RCC1. The N-terminal region of RCC1 is highly basic and similar to that of the N-terminal tail of H3. RCC1 is responsible for catalyzing the formation of RanGTP, which is essential for mitotic spindle assembly and nuclear envelope formation. Rare PTMs of RCC1 were identified and various biochemical experiments showed that inhibition of these modifications caused mitotic defects.; Lastly, we were interested in understanding the mechanisms involved in embryonic stem (ES) cell differentiation, the process that defines specialized cells. During the differentiation process, many genes are destined to be turned "on" or "off'. Histone PTMs and their associated proteins are implicated in ES cell differentiation. We identified a proteolyically cleaved H3 protein associated with cell differentiation. MS analyses identified the specific sites of cleavage and characterized the PTMs on the cleaved H3 protein.