The molecular mechanisms that regulate protein function via post translational modifications (PTMs) are largely unknown. Among the amino acid residues that provide scaffolds for PTMs, cysteine is rapidly becoming appreciated for its ability to host numerous modifications. Depending on the type and location of the cysteine based PTMs, differential effects on protein localization, function or stability can be achieved. We have previously developed methods to identify endogenous cysteine PTMs. To date 2,596 sites of cysteine PTMs have been mapped to 1,838 proteins. We aim to investigate techniques to improve the mass spectrometric identification of cysteine residues in the mouse liver proteome. Mass spectrometry techniques can only detect peptide fragments within a specific size range, limiting our ability to identify modified cysteine residues. In order to potentially increase the protein sequence coverage, we compared the fidelity of the endoproteinases Trypsin, Asp-N and Lys-C for use during enrichment of cysteine PTMs. The use of Trypsin resulted in the identification of 1,314 cysteines. Protein digestion with the alternate enzymes Asp-N and Lys-C resulted in greater sequence coverage and detection of 440 additional cysteine residues, which represents a 25% increase in coverage. This is paramount to gaining a complete understanding of the biochemical and biological processes in which these modifications occur.

Improved protein digestion for the mass-spectrometric detection of cysteine palmitoylation

249th National Meeting (2015)

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