Performance Comparison of Three Trypsin Columns Used in Liquid Chromatography

Overview

Journal J Chromatogr A

Publisher Elsevier

Specialty Chemistry

Date 2017 Feb 21

PMID 28215403

Citations 6

Authors

Tereza Slechtova

Martin Gilar

Kveta Kalikova

Stephanie M Moore

James W Jorgenson

Eva Tesarova

Affiliations

Soon will be listed here.

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 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N-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.

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