A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots
| Autor: | Leon E. Niezen, Sam Wouters, Thalassa S.E. Valkenburg, Garry L. Corthals, Irena Dapic, Sebastiaan Eeltink, Bert Wouters, Peter J. Schoenmakers |
|---|---|
| Přispěvatelé: | Supramolecular Separations (HIMS, FNWI), Chemical Engineering and Industrial Chemistry, Faculty of Engineering, Centre for Molecular Separation Science & Technology, Chemical Engineering and Separation Science |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Electrophoresis
Proteome Immobilized enzyme Protein digestion Polymers Clinical Biochemistry 02 engineering and technology 01 natural sciences Biochemistry Sample preparation in mass spectrometry bottom-up Separation Analytical Chemistry Capillary Digestion (alchemy) Porous Polymer Monolith medicine Humans Trypsin Denaturation (biochemistry) mass spectrometry Chromatography Chemistry 010401 analytical chemistry Organic Chemistry Proteolytic enzymes Cycloparaffins Polymer monolith General Medicine Blood Proteins Microfluidic Analytical Techniques 021001 nanoscience & nanotechnology Enzymes Immobilized Blood proteins 0104 chemical sciences Microreactor Molecular Medicine Digestion Proteolytic Digestion Dried Blood Spot Testing LIQUID-CHROMATOGRAPHY 0210 nano-technology medicine.drug |
| Zdroj: | Journal of Chromatography A, 1491, 36-42. Elsevier |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/j.chroma.2017.01.078 |
| Popis: | 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 240 kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5 min 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 22.5 h 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. (C) 2017 The Authors. Published by Elsevier B.V. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect