title: Native protein proteolysis in an immobilized enzyme reactor as a function of temperature.
abstract: 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 on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced 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.
authors: Department of Chemistry, 560 Oval Drive, Purdue University, West Lafayette, Indiana 47906, USA.
authors: Dinelia
authors: D
authors: Rivera-Burgos
authors: Fred E
authors: FE
authors: Regnier
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019149:Bioreactors; D004220:Disulfides / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004220:Disulfides; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D014357:Trypsin