Abstract
Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.
Highlights
Trypsin is the protease of choice for protein sample digestion in proteomics
MS-based data on peptides are searched against amino acid sequence databases, which benefits from the relative stringent cleavage specificity of trypsin as the search algorithms incorporate the cleavage rule as a filtering criterion
The analysis showed that about 90 % of the detected peptides with missed cleavage sites could be attributed to the following sequence motifs: (1) Arg or Lys with a neighboring proline at the C-terminal side, (2) two successive basic residues (Arg-Arg, Arg-Lys, Lys-Arg, Lys-Lys), and (3) Arg or Lys with an aspartic acid or glutamic acid residue at either N-terminal or C-terminal side
Summary
A serine protease, is commonly used as an important enzymatic reagent in biochemistry and biology It is almost indispensable especially for the digestion of protein samples to peptides in bottom-up proteomics [1]. An average length of tryptic peptides is 14 amino acids This number has been deduced from an in silico digestion of human proteins in the UniProt database [1]. Because of this reasonable size as well as the presence of a positive charge at the C-terminal Arg or Lys, which enhances the ionization process in the positive ionization mode, tryptic peptides are highly amenable to mass spectrometric measurements. There are at least two defined positive charges in tryptic peptides (at both Nand C-termini), which is favorable for a good fragmentation in MS/MS analyses [1,2]
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