A microfluidic reactor for rapid, low‐pressure proteolysis with on‐chip electrospray ionization

Abstract

A microfluidic reactor that enables rapid digestion of proteins prior to on-line analysis by electrospray ionization mass spectrometry (ESI-MS) is introduced. The device incorporates a wide (1.5 cm), shallow (10 microm) reactor 'well' that is functionalized with pepsin-agarose, a design that facilitates low-pressure operation and high clogging resistance. Electrospray ionization is carried out directly from a short metal capillary integrated into the chip outlet. Fabrication, involving laser ablation of polymethyl methacrylate (PMMA), is exceedingly straightforward and inexpensive. High sequence coverage spectra of myoglobin (Mb), ubiquitin (Ub) and bovine serum albumin (BSA) digests were obtained after <4 s of residence time in the reactor. Stress testing showed little loss of performance over approximately 2 h continuous use at high flow rates (30 microL/min). The device provides a convenient platform for a range of applications in proteomics and structural biology, i.e. to enable high-throughput workflows or to limit back-exchange in spatially resolved hydrogen/deuterium exchange (HDX) experiments.

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

“…Microchip-based IMERs are very important to the development of fully integrated, high-throughput microfluidic systems containing protein digestion, peptides separation and identification [5,12,30]. Based on the hydrophilic monolith that can be easily and quickly prepared in the specified position of microchannels, microchip-based IMERs were prepared and integrated with nanoRPLC-ESI-MS/MS via a packed electrospray emitter tip.…”

“…To solve these problems, recently much attention has been paid to the development of immobilized enzyme reactors (IMERs), which not only have high digestion efficiency, but also can be easily coupled with separation and detection systems to achieve automated and high-throughput protein analysis [5][6][7]. Furthermore, with the development of nanoscale reversed phase liquid chromatography coupled with mass spectrometry (nanoRPLC-MS) and chip-MS, IMERs prepared in capillaries and on microchips are highly desirable for the construction of integrated platforms involving online protein digestion, peptides separation and identification [8][9][10][11][12].…”

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

“…Digestion usually occurs after a protease such as trypsin is mixed with substrate proteins in solution. However, this strategy requires low enzyme concentrations to restrict self-digestion of the protease, so digestion times are generally 1 h or more (91 reactors with proteases immobilized on solid supports including monoliths (92,93), membranes (94,95), polymeric microfluidic channels (94,96), and resins (97,98). With thicknesses of only 10-200 μm, membranes provide a unique platform to control protein digestion.…”

Time-resolved electrospray mass spectrometry — a brief history

“…1c). 32,33 Briefly, HDX relies on the ability of backbone amide hydrogens to undergo exchange with deuterium atoms in solution. 34,35 At physiological pH, the exchange is base-catalyzed although acid catalysis becomes dominant below pH 2.6.…”