Abstract
Trypsin is a protease, which is commonly used for the digestion of protein samples in proteomic experiments. The process of trypsin autolysis is known to produce autolytic peptides as well as active enzyme forms with one or more intra-chain splits. In consequence, their variable presence can influence the digestion of a protein substrate in the reaction mixture. Besides two major and well-studied forms named β-trypsin and α-trypsin, there are also other active trypsin forms known such as γ-trypsin and pseudotrypsin (ψ-trypsin). In this work, the cleavage specificity of ψ-trypsin was evaluated using in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the resulting peptides. The numbers of produced and matching peptides were similar to those obtained using α-/β-trypsin. The same experience was obtained with a real complex protein sample from rat urine. In previous reports, ψ-trypsin was supposed to generate non-specific cleavages, which has now been reevaluated. Purified ψ-trypsin cleaved all analyzed proteins preferentially on the C-terminal side of Lys and Arg residues in accordance with the canonical tryptic cleavage. However, a minor nonspecific cleavage performance was also registered (particularly after Tyr and Phe), which was considerably higher than in the case of trypsin itself.
Keywords: Autolysis, chymotrypsin, in-gel digestion, MALDI-TOF MS, pseudotrypsin, trypsin.
Graphical Abstract
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