title: Insight into Trypsin Miscleavage: Comparison of Kinetic Constants of Problematic Peptide Sequences.
abstract: Trypsin, a high fidelity protease, is the most widely used enzyme for protein digestion in proteomic research. Optimal digestion conditions are well-known and so are the expected cleavage products. However, missed cleavage sites are frequently observed when acidic amino acids, aspartic and glutamic acids, are present near the cleavage site. Also, the sequence motifs with successive lysine and/or arginine residues represent a source of missed cleaved sites. In spite of an adverse role of missed cleaved peptides on proteomic research, the digestion kinetics of these problematic sequences is not well-known. In this work, synthetic peptides with various sequence motifs were used as trypsin substrates. Cleavage products were analyzed with reversed-phase high performance liquid chromatography, and the kinetic constants for selected missed cleavage sites were calculated. Relative digestion speed for lysine and arginine sites is compared, including the digestion motifs flanked with aspartic and glutamic acid. Our findings show that DK and DTR motifs are cleaved by trypsin with 3 orders of magnitude lower speed than the arginine site. These motifs are likely to produce missed cleavage peptides in protein tryptic digests even at prolonged digestion times.
authors.affiliation: †Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43, Prague, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Šlechtová
authors.affiliation: ‡Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.affiliation: †Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43, Prague, Czech Republic.
authors.forename: Květa
authors.initials: K
authors.lastname: Kalíková
authors.affiliation: †Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43, Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Tesařová
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D014357:Trypsin