Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/42098
Title: A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots.
Authors: Wouters, B.
Dapic, I.
Valkenburg, Thalassa S.E.
Wouters, S.
Niezen, L.
Eeltink, S.
Corthals, G. L.
Schoenmakers, P. J.
First Published: 28-Jan-2017
Publisher: Elsevier
Citation: Journal of Chromatography A, 2017, 1491, pp. 36-42
Abstract: A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character.
DOI Link: 10.1016/j.chroma.2017.01.078
ISSN: 0021-9673
eISSN: 1873-3778
Links: https://www.sciencedirect.com/science/article/pii/S0021967317301759?via%3Dihub
http://hdl.handle.net/2381/42098
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © the authors, 2017. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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