Performance comparison of three trypsin columns used in liquid chromatography⋆
| Autor: | Martin Gilar, Stephanie Moore, James W. Jorgenson, Květa Kalíková, Eva Tesařová, Tereza Šlechtová |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Autolysis (biology) Sorbent Hydrochloride Protein digestion 01 natural sciences Biochemistry Article Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound medicine Trypsin chemistry.chemical_classification Chromatography 030102 biochemistry & molecular biology Chemistry 010401 analytical chemistry Organic Chemistry General Medicine Enzymes Immobilized 0104 chemical sciences Enzyme Covalent bond Performance comparison medicine.drug Chromatography Liquid |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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