Transpeptidation during the analytical proteolysis of proteins.
Since peptide mapping with proteolytic enzymes such as trypsin and Staphylococcus aureus V8 protease is a powerful tool for the characterization of proteins, investigators should be cognizant of possible artifacts due to the technique itself. This article describes the identification of minor peaks found in the maps of recombinant human relaxin and insulin-like growth factor I as transpeptidation products. Both proteins have some homology to insulin with relaxin being composed of two chains designated A and B, while insulin-like growth factor I is composed of a single polypeptide chain. Digestion of relaxin with trypsin at pH 7.2 yields two peptides, T2,3(A10-18) and T7(B10-13), linked together by a disulfide bond. An unexpected component at a 10% level was identified to be the T2-T7 peptide pair where T3(ArgA18) has formed a peptide bond with the amino-terminal LeuB10 of the T7 peptide. It was also observed that the digestion of insulin-like growth factor I with V8 protease normally yields two peptides V4(13-20) and V9(59-70) linked by a disulfide bridge. A minor peak at a 1 to 2% level was identified to be a single polypeptide resulting from the formation of a peptide bond between the amino-terminal Met59 of V9 and the carboxyl-terminal Asp20 of V4, with the disulfide bond intact. These transpeptidation products were isolated by reversed-phase HPLC and identified using amino-terminal sequence and mass spectrometric analyses.
Canova-Davis
E
E
Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Kessler
T J
TJ
Ling
V T
VT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012065:Relaxin / Q000737:chemistry*; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D012065:Relaxin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin