Advances in protein proteolysis relating to proteomics

Dinelia Rivera-Burgos, Purdue University


Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few min have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin-IMER can in fact digest transferrin in a few min; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin-IMER produces i) numerous peptides bearing multiple lysine and/or arginine residues and ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.^ Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies based on its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when non-reduced transferrin was digested at 60°C. Large numbers of missed cleavages were observed from species regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and non-reduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis. ^




Fred E. Regnier, Purdue University.

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