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.

On-line microfluidic immobilized-enzyme reactors: A new tool for characterizing synthetic polymers

“…Hydrodynamic chromatography analysis indicated that the particles had an average size of 130 nm (see Supplementary Material, section S-2 for the corresponding data). Based on previous information we expect the diameter of the through pores of the IMER-containing monoliths to be in the range of 0.8e1.5 mm [18]. For recording the calibration curves, 5000 ppm solutions of all standards were individually prepared in unstabilized THF and diluted to 1000 ppm, also in unstabilized THF, for analysis (see Supplementary Material, section S-3 for the calibration curves).…”

“…The procedure to immobilize enzymes in-situ in the microfluidic channels was adapted from the method described elsewhere [18], and consisted of three major steps: i) surface modification of the cyclic-olefin copolymer microchannel and in-situ polymerization of the polymer monolith, ii) photografting of poly(ethylene glycol) methacrylate and 2-vinyl-4,4-dimethylazlactone, and iii) lipase immobilization. During the last step, a 2000 mg/L aqueous solution of lipase containing NaCl (0.9% by weight) was pumped through the microdevice for 2 h at 0.5 mL min À1 .…”

“…In the context of proteomics studies, immobilized-enzyme reactors (IMER) have been applied for the (on-line) digestion of proteins. Through the immobilization of proteases such as trypsin, digestion times were reduced to seconds or minutes [18]. IMERs are more efficient for protein digestion than in-solution enzymatic digestion, because i) mass transfer between enzymes and substrate molecules occurs faster as the diffusion distances between the molecules are reduced and ii) the increased enzyme concentration in the reactor, which is only possible because autodigestion of the enzymes is prevented by their immobilization and, thus, isolation from each other.…”

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

“…To cope with the increasing numbers of samples and to implement the technique in a QC environment with HRAM LC-MS, sample preparation reproducibility is also required which can be provided by automation through online digestion or robotic systems [13][14][15][16][17].…”

Kinetic Analysis of Enzymes Immobilized in Porous Film Arrays

“…Enzyme immobilization on solid supports has fueled advances in biosensing, 1,2 biocatalysis, 3 and proteomics, 4,5 among other fields. The history, applications, and available methods of enzyme immobilization are vast, as covered in various reviews.…”