![\"trypsin](\"http:\/\/admin.acceleratingscience.com\/wp-content\/uploads\/2014\/05\/trypsin.jpg\")
Researchers have often used trypsin to digest protein samples prior to mass spectrometric proteomic analysis, but they may not realize that trypsin may also be used to tag peptides for analysis. <\/p>\nResearchers have often used trypsin to digest protein samples prior to mass spectrometric proteomic analysis, but they may not realize that trypsin may also be used to tag peptides for analysis. <\/p>\n
Trypsin is a dual-action enzyme dependent upon reaction conditions. Under the sample preparation conditions commonly associated with protein digestion prior to analysis via mass spectrometry (MS), trypsin acts as a protease, chopping proteins into convenient and consistent fragments at precise digestion sites. In the presence of organic solvents such as ethanol, however, it turns into a ligase, attaching suitable tags onto the N-termini of digested proteins via the formation of a new peptide bond.<\/p>\n
Pan et al. (2014) report a new enzymatic method for N-terminal labeling that exploits this dual activity, using stable biotinylated tags that can be used to clean up the sample prior to MS analysis.1<\/span><\/sup> In exploring this method, their intention was to develop a terminal labeling technique that did not modify side chains or disturb post-translational modifications on lysine residues. Current chemical labeling strategies can interfere with these factors or be indistinguishable from in vivo<\/em> events. The research team also wanted to develop a less expensive alternative to stable isotope labeling by amino acids in cell culture (SILAC) that could be used in vitro<\/em>.<\/p>\n First, the researchers biotinylated 12<\/sup>C6<\/sub>-L-arginine-OEt and 13<\/sup>C6<\/sub>-L-arginine-OEt to construct stable isotope coded affinity tags; these were then esterified, forming light and heavy stable biotin-R-OEt tags, respectively. They confirmed the success of this reaction by matrix-assisted laser desorption\/ionization–time-of-flight (MALDI-TOF) analysis.<\/p>\n Labeling reactions were tested using a synthetic peptide, ALKAADTIGYPVMIR, with a single trypsin cleavage site. Following trypsin digestion, the fragments were incubated with the biotin tags in the presence of trypsin and ethanol. Pan et al. confirmed successful N-terminal labeling by MALDI-TOF analysis in the primary synthetic peptide, as well as in two further, similarly constructed, peptides. The researchers found that the labeling reaction did not go to completion, however, leaving a majority of fragments unlabeled<\/span>.<\/p>\n