title: 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 300Å pores, packed in 2.1×30mm 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.
authors.affiliation: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague, Czechia.
authors.forename: Tereza
authors.initials: T
authors.lastname: Šlechtová
authors.affiliation: Waters Corporation, 34 Maple Street, Milford, MA 01757, USA. Electronic address: Martin_Gilar@waters.com.
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.affiliation: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague, Czechia.
authors.forename: Květa
authors.initials: K
authors.lastname: Kalíková
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Moore
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA.
authors.forename: James W
authors.initials: JW
authors.lastname: Jorgenson
authors.affiliation: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague, Czechia.
authors.forename: Eva
authors.initials: E
authors.lastname: Tesařová
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D014357:Trypsin
keywords: Activity; Digestion efficiency; Immobilized enzymatic reactor; Trypsin