title: Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin.
abstract: 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 process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes 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 minutes; 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.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47906, USA.
authors.forename: Dinelia
authors.initials: D
authors.lastname: Rivera-Burgos
authors.forename: Fred E
authors.initials: FE
authors.lastname: Regnier
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D004063:Digestion; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D059748:Proteolysis; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D014168:Transferrin; D014357:Trypsin