Endoproteinases Asp-N and Glu-C have been used for protein characterization for over 30 years and have gained importance recently due to advancements in mass spectrometry techniques. Asp-N preferentially cleaves proteins at the N-terminus of aspartic and cysteic acid (1–3). Glu-C cleaves at the C-terminus of glutamic and aspartic residues (4–6). Due to their specific cleavage sites, these proteinases create unique peptide fragments available for mass spectrometry analysis. Comparing protein sequences or mapping data from Asp-N or Glu-C digests to that of other proteinases promotes higher confidence in data (7–12).
Protein digestion is required for either the bottom-up or middle-down approach to protein analysis. In the bottom-up approach, the optimal peptide size range for analysis is 600–5,000Da. Recent advancements in mass spectrometry such as electron transfer dissociation (ETD) and electron capture dissociation (ECD), the potential to combine with collision activated dissociation (CAD) and in software, have expanded the peptide size range to 600–20,000Da. These advancements have promoted the middle-down approach to protein analysis (9, 13–16). However, some peptides still may fall above or below the desired peptide size range. This results in decreased protein coverage and incomplete data collection.
To increase protein coverage, additional proteinases have been used. Using an alternative proteinases individually or in combination with other proteinases creates a unique peptide map that may include sequences not seen in trypsin digestions. Overlaying peptides obtained from digestion with alternative proteinases increases protein coverage and overall confidence in protein identification. When alternative enzymes are used, larger peptides are cleaved into smaller fragments, which are more manageable for the instrumentation. Conversely, protein sections cleaved into peptides too small for analysis by one enzyme might be cleaved into larger peptides with a different enzyme. Alternative proteinases also help to overcome incomplete digestion caused by PTMs, which prevent the proteinases from accessing that particular site. By using alternative enzymes, the protein might be cleaved at sites further away from the PTM. The examples above show that alternative proteinases are beneficial to protein analysis (9, 13–16).