Revisiting the effect of trypsin digestion buffers on artificial deamidation

Deamidation of asparagine and glutamine residues occurs spontaneously, influenced by pH, temperature, and incubation time, and can be accelerated by adjacent amino acid residues. Incubation conditions used for proteolytic digestion in bottom-up proteomic studies can induce significant deamidation. This has prompted studies into modifications to common trypsin digestion protocols, including shorter incubation times and specific lysis buffers, to minimize chemical deamidation. Prior work suggested ammonium acetate at pH 6 to minimize chemical deamidation, but this buffer has compatibility issues with trypsin digestion and common assays (e.g., bicinchoninic acid assays). Here, we re-evaluated former comparisons of Tris-HCl, ammonium bicarbonate, and triethylammonium bicarbonate buffers for the amount of artificial, chemically induced deamidation generated in a standard bottom-up proteomics workflow and added an evaluation of two commonly used and biologically compatible buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and EPPS, (3-[4-(2-Hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid). Our findings show that HEPES exhibited the least amount of artificial deamidation and is a wise choice for general proteomic experiments, especially for studies considering N-glycosylation.