A key component in any bottom-up (shotgun) proteomics workflow is the enzymatic digestion of proteins into peptides prior to analysis by liquid chromatography coupled to mass spectrometry (LC-MS). Trypsin is the most widely used enzyme due to its stringent specificity for cleavage at the C-terminal of lysine and arginine, creating positively charged peptides compatible for analysis by tandem mass spectrometry. A robust and efficient (i.e. complete) digestion protocol is essential for reliable, accurate peptide identification and quantification, as well as the identification of genuine post-translation modifications (i.e., those not induced during sample preparation). Previously, it has been demonstrated that incubation conditions during trypsin digestion including temperature, extraction buffer components and pH can induce artifactual chemical modifications.
Using a ‘simple’ system (bovine serum albumin, BSA) and two complex samples (mouse brain tissue lysate and E. coli K-12 whole cell lysate), we evaluated the effects of trypsin digestion carried out at 4 °C and 37 °C for 4 and 16 hours (overnight). Preliminary data indicated that tryptic digestion of proteome extracts with the traditional digestion protocol of 37 °C overnight results in increased artifactual modifications such as deamidation of asparginine and glutamine. Despite having the least number of tryptic miscleavages, digestion at 37 °C overnight had significantly lower protein sequence coverage compared to digestion at 4 °C overnight and 4 hours digestion at 4 °C and 37 °C. No significant differences were observed in the number of proteins identified under different incubation times and temperatures, with the exception of 4 hours digestion at 4 °C which was significantly lower. Next steps will include exploring the effects of the different incubation times and temperatures on additional heat-labile or heat-induced chemical modifications.