Recognition of trypsin autolysis products by high-performance liquid chromatography and mass spectrometry

• Published in: Analytical chemistry (Washington) Vol. 62; no. 21; pp. 2391 - 2394
• Main Authors: Vestling, Martha M, Murphy, Constance M, Fenselau, Catherine
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-11-1990
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Autolysis - metabolism; Biological and medical sciences; Cattle; Chromatography, High Pressure Liquid - methods; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Mass Spectrometry - methods; Molecular Sequence Data; Swine; Trypsin - metabolism; Bovine; Enzyme; Edman degradation; HPLC chromatography; Pig; Autolysis; Vertebrata; Trypsin; Mammalia; Peptide fragment; PH; Artiodactyla; Pancreas; Mass spectrometry; Ungulata; Aminoacid sequence
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac00220a025

Potential artifactual contributions are assessed in high-pressure liquid chromatograms and fast atom bombardment mass spectra from autolysis of different preparations of the widely used protease trypsin. Both commercially supplied and laboratory-purified samples were examined. Bovine pancreatic trypsin (1 mg/mL) was found to be completely destroyed in 2 h at pH 8.5, degraded to a complex mixture of small peptides which were characterized by their molecular weights. Some identifications were supported by sequencing by tandem mass spectrometry or by mass spectrometric analysis of the mixture resulting from a single Edman degradation. Autolysis of porcine pancreatic trypsin produced a completely different set of peptides. Five sites of hydrolysis at asparagine residues in bovine trypsin were also identified.