title: A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots.
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.
authors.affiliation: University of Amsterdam, van't Hoff Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands; TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands. Electronic address: b.wouters@uva.nl.
authors.forename: Bert
authors.initials: B
authors.lastname: Wouters
authors.affiliation: University of Amsterdam, van't Hoff Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Irena
authors.initials: I
authors.lastname: Dapic
authors.affiliation: University of Leicester, Department of Chemistry, University Road, Leicester LE1 7RH, United Kingdom.
authors.forename: Thalassa S E
authors.initials: TS
authors.lastname: Valkenburg
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Sam
authors.initials: S
authors.lastname: Wouters
authors.affiliation: University of Amsterdam, van't Hoff Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Leon
authors.initials: L
authors.lastname: Niezen
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Eeltink
authors.affiliation: University of Amsterdam, van't Hoff Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Garry L
authors.initials: GL
authors.lastname: Corthals
authors.affiliation: University of Amsterdam, van't Hoff Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schoenmakers
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D003516:Cycloparaffins / Q000737:chemistry*; D059788:Dried Blood Spot Testing / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D011108:Polymers / Q000737:chemistry*
chemical_list: D001798:Blood Proteins; D003516:Cycloparaffins; D004800:Enzymes, Immobilized; D011108:Polymers
keywords: Bottom-up; Digestion; Mass spectrometry; Polymer monolith; Proteome; Trypsin