The use of proteases complementary to trypsin to probe isoforms and modifications

Abstract

The wide diversity of proteins expressed in a cell or a tissue as a result of gene variants, RNA editing or PTMs results in several hundred thousand distinct functional proteins called proteoforms. The large-scale analysis of proteomes has been driven by bottom-up MS approaches. This allowed to identify and quantify large numbers of gene products and perform PTM profiling which yielded a significant number of biological discoveries. Trypsin is the gold standard enzyme for the production of peptides in bottom-up approaches. Several investigators argued recently that the near exclusive use of trypsin provided only a partial view of the proteome and hampered the discovery of new isoforms. The use of multiple proteases in a complementary fashion can increase sequence coverage providing more extensive PTM and sequence variant profiling. Here the various approaches to characterize proteoforms are discussed, including the use of alternative enzymes to trypsin in shotgun approaches to expand the observable sequence space by LC-MS/MS. The technical considerations associated with the use of alternative enzymes are discussed.

Biotechnological Applications of Proteases in Food Technology

“…Trypsin, either pancreatic porcine or bovine, is the family of proteases most broadly utilized in the hydrolysis of protein samples for proteome analysis, useful due to its high specificity. This way, the treatment can release specific and “standard” peptide fragments that enable to achieve “peptide maps” (Giansanti and others ; Trevisol and others ). This allows the identification of the samples under study.…”

“…Trypsin hydrolysis of proteins also has the advantage of producing not very long peptides with basic C‐terminus, which are suitable for collision‐induced dissociation (CID) tandem mass spectrometric analysis (Stosova and others ; Tsiatsiani and others ). Other sources of trypsin‐like or even other enzymes have been considered to be used in sample preparation for proteomic studies (Wang and others ; Trevisol and others ). Kiser and others () suggested trypsin from Streptomyces erythraeus as a candidate for its utilization in proteomic analysis, because it is more resistant to urea action, significantly more active, and much more resistant to autolysis than bovine trypsin.…”

Sample preparation in foodomic analyses

“…It has to be repeated that trypsin is still “the gold standard protease” for protein digestion before LC‐MS/MS analysis, and this enzyme is almost exclusively used for proteomic analyses of food samples . However, MS‐based proteomic analysis still cannot access all proteome components since trypsin sequence specificity may not be suitable for detection . Increased proteome coverage was observed by combination of several proteases and cleavage strategies .…”

“…One of already evaluated alternatives for application in shotgun bottom‐up proteomics is LysargiNase . This enzyme cleaves proteins at the N‐terminal side of Arg and Lys, and compared to tryptic peptides, cleavage products contain two protons that are positioned at the N‐terminus.…”

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

“…However, it is not known if these PTMs are conserved in HTT expressed in Sf9 insect cells. Purified HTT Q23 and Q54 from Sf9 and EXPI293F were subjected to bottom-up proteomics (24,25). PTMs were mapped for HTT expressed in Sf9 and EXPI293F cells and compared with published PTMs of mammalian derived HTT (Tables 1-4 detail results for HTT Q23 samples from Sf9 and EXPI293F production, complete data can be found on PRIDE (accession PXD010865) and in Zenodo https://zenodo.org/record/2169035).…”