Performance comparison of three trypsin columns used in liquid chromatography

Abstract

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 Å pores, packed in 2.1 × 30 mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37 °C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics

“…Enzymatic digestion of proteins is a key sample preparation step in bottom-up proteomics workflows. The efficiency of protein digestion is influenced by a number of factors including the quality of the enzymes, temperature, incubation time, buffer composition, including pH, and volume of digestion buffer . A number of studies have devoted resources to increasing the efficiency of enzymatic digestion and thereby reducing levels of peptide miscleavage events and improving the quantitative accuracy and reproducibility of LC-MS measurements. ,− We determined the optimal bead and enzyme amounts for each protein amount contained within a certain volume of protein lysate based on the number of unique sequences identified and the average miscleavage rates (Supporting Information Figure 5).…”

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein

“…These IMERs also have the advantage to be reusable and easily automated. Numerous IMERs based on the immobilization of trypsin [4,[16][17][18][19][20], and to a lesser extent of pepsin [15,21,22], have been reported in the literature. Yet, the focus was set on the identification of proteins and few information was given on PTMs.…”

Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated μSPE reactors and mass spectrometry

“…The same crosslinking chemistry was used to immobilise trypsin onto the generic CMD μSPEed cartridge. The immobilised trypsin activity and repeatability within the cartridge was evaluated using BAEE as a model compound [ 31 , 32 ]. In the presence of trypsin, BAEE produced N α -benzoyl-l-arginine (BA) and ethanol via cleavage at the carboxyl side of arginine as seen in Fig.…”