A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies

Abstract

Mass spectrometry (MS) coupled to sample preparation and separation techniques has become a primary tool for proteomics studies. However, due to sample complexity, it is often challenging to achieve fast and efficient sample preparation prior to MS analysis. In recent decades, monolithic materials have been developed not only as chromatographic media, but also as efficient solid supports for immobilizing multiple types of affinity reagents. Herein, the N-acryloxysuccinimide-co-acrylamide-co-N,N'-methylenebisacrylamide (NAS-AAm-Bis) monolith was fabricated within silanized 200 μm i.d. fused-silica capillaries and was used as an immobilized enzyme reactor (IMER). The column was conjugated with trypsin/Lys-C and Lys-N enzymes to allow enzymatic digestions to occur while protein mixture was loaded onto the IMER column followed by MS-based proteomics analysis. Similar MS signal and protein sequence coverage were observed using protein standard bovine serum albumin (BSA) compared to in-solution digestion. Furthermore, mouse serum, yeast, and human cell lysate samples were also subjected to enzymatic digestion by both IMER (in seconds to minutes) and conventional in solution digestion (overnight) for comparison in large-scale proteomics studies. Comparable protein identification results obtained by the two methods highlighted the potential of employing NAS-based IMER column for fast and highly efficient sample preparation for MS analysis in proteomics studies.

Characterization of an immobilized enzyme reactor for on-line protein digestion

“…The IMER had 200 positive values where it produced higher coverage and there was one instance where the coverage was identical resulting in a value of zero. The IMER digestion presented in this study was faster than previous reports for on-line yeast cell lysate digestion [21, 46, 47] and identified approximately the same number of yeast proteins (541 reported in Feng, et al [21] compared to 507 found in this study). It is important to note that the sample used in this study was the insoluble portion of a yeast cell lysate, containing approximately 60% proteins that are identified as membrane-associated.…”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…On‐line sample trypsinization can be performed in seconds using an IMER microreactor . Additional, increase of efficacy can be achieved by co‐immobilization with Lys‐C . With a new monolithic bioreactor with 3D‐printed interface stationary phase, high concentration of protease activity can be achieved .…”

Immobilized enzyme reactors based on monoliths: Effect of pore size and enzyme loading on biocatalytic process

“…Moreover, the advantages of methacrylate‐based monolithic matrices include mechanical and chemical stability, as well as the possibility of synthesis of these materials in various geometrical formats, namely, in the shape of disks, columns or tubes . Owing to the mentioned advantages of monoliths, they have become popular not only for application in different kinds of chromatography and solid‐phase extraction , but also for the preparation of flow‐through immobilized enzyme reactors (IMERs) suitable for proteomics , microfluidic analysis , pharmaceutics , biodiesel and oligosaccharides production, etc. In most cases, the covalent binding of enzyme to the surface of macroporous monoliths was applied .…”