Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface

Abstract

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 µm wide, 150 µm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40–60 µm diameter beads. Separation was performed in channels etched 10 µm deep, 30 µm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 µm i.d. and 180 µm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 µL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 µL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3–6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10–15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications. Copyright © 2000 John Wiley & Sons, Ltd.

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

“…This system integrates steps for proteomic analysis using MALDI-TOF MS such as digestion, mixing with a matrix solution, and depositing onto a MALDI target plate. Compared with several reported microfluidic devices for protein digestion that require reaction times of 3 to 10 min to achieve the desired sequence coverage [26,43,49,51], our automated digestion system with matrix mixing and spotting affords comparable coverage after a residence time of less than 1 min. In addition, the open channel format obviates the need for producing 3-D supports in the reactor or packing the reactor with beads.…”

“…Strategies for identifying proteins focus on accurate, sensitive, simple, and high throughput analyses achieved by either reducing processing time or with multi-channel devices [23,24]. Trypsin digestion for protein sample preparation is the most frequently used step in proteomic analysis due to the robust nature of this enzyme and the extensive databases and software tools for trypsin digests of proteins [25,26]. However, the long sample incubation times required for trypsin digestion in solution and the extensive sample treatment steps result in long protein processing times.…”

“…Typically, the reaction time required for homogeneous solution tryptic digestions is tens of hours to achieve a sufficient population of peptide fragments for effective PMF because the enzyme-to-substrate ratio must be kept low to avoid interferences from autodigested trypsin [26]. To overcome this problem, enzymeimmobilization on solid supports for solid-phase bioreactors has been introduced [26,[31][32][33].…”

“…Compared with homogeneous reactors, solid-phase enzymatic bioreactors offer several advantages such as faster and simpler sample preparation steps, fewer enzyme autolysis products, good reproducibility, larger enzyme-tosubstrate molar ratios, high digestion efficiency, and the possibility of repeated use. Wang et al [26] developed a microfluidic chip that was packed with trypsin-loaded beads in a fluidic channel. It was found that the bead-packed chip gave faster protein digestion and fewer trypsin autolysis products compared with a homogeneous reaction.…”

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

“…134,135 Aside from their potential to perform sample-handling procedures in minute volumes, they dispense with the long incubation times required for digestions because of increased reaction kinetics in low sample volumes. 135 Wang et al 136 described a microfluidic device that integrated a protein digestion bed on a monolithic surface using trypsin immobilized on beads. The flow rate at which the substrate solution was pumped through the reactor bed determined the rate and efficiency of digestion.…”