Thiol-ene Monolithic Pepsin Microreactor with a 3D-Printed Interface for Efficient UPLC-MS Peptide Mapping Analyses

• Published in: Analytical chemistry (Washington) Vol. 89; no. 8; pp. 4573 - 4580
• Main Authors: Jönsson, Alexander, Svejdal, Rasmus R, Bøgelund, Nanna, Nguyen, Tam T. T. N, Flindt, Henrik, Kutter, Jörg P, Rand, Kasper D, Lafleur, Josiane P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18-04-2017
• Subjects: Analytical chemistry; Chips; Chromatography; Digestion; Efficiency; Enzymes; Mass spectrometry; Pepsin; Peptides; Proteins; Reactors
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.6b05103

To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.