Development of an open-tubular trypsin reactor for on-line digestion of proteins.
ABSTRACT: A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm(-2) trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol microL(-1) horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm x 50 microm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.
Project description:Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N?-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.
Project description:Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20??m inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20??m ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10??m ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.
Project description:Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.
Project description:We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and ?-casein as model proteins.
Project description:Immobilized trypsin produces very fast protein digestion, which is attractive for application to high throughput bottom-up proteomics. While there is a rich literature on the preparation of immobilized trypsin, there are very few studies that investigate its application to complex proteomic samples. In this work, we compared solution-phase trypsin with trypsin immobilized on magnetic microspheres for digestion of two complex proteomes, Escherichia coli and the MCF7 cell line. The digests were separated by HPLC, and detected with a Q-Exactive mass spectrometer, which generated high resolution and high quality parent- and fragment-ion mass spectra. The data were analyzed using MaxQuant. We make several conclusions about the features of immobilized trypsin digestion of complex proteomes. First, both immobilized and solution-phase trypsin generate peptides that sample the same protein pool. Second, immobilized trypsin can digest complex proteomes two orders of magnitude faster than solution-phase trypsin while retaining similar numbers of protein identifications and proteome depth. Digestion using immobilized trypsin for 5-min produces a similar number of missed cleavages as solution-based trypsin digestion for 4-h; digestion using immobilized trypsin for 20-min produces a similar number of missed cleavages as solution-based trypsin digestion for 12-h. Third, immobilized trypsin produces quantitatively reproducible digestion of complex proteomes. Finally, there is small but measurable loss of peptide due to non-specific adsorption to the immobilization matrix. This adsorption generates a bias against detection of basic peptides.
Project description:A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ?1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.
Project description: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 ?m 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.
Project description:BACKGROUND: Lignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy. Thermochemical pretreatment processes have been developed to address the challenge of biomass recalcitrance, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale. In this study, we demonstrate that the type of reactor in which pretreatment is carried out can profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose. RESULTS: Multi-scale microscopy and quantitative image analysis was used to investigate the impact of different biomass pretreatment reactor configurations on plant cell wall structure. We identify correlations between enzymatic digestibility and geometric descriptors derived from the image data. Corn stover feedstock was pretreated under the same nominal conditions for dilute acid pretreatment (2.0 wt% H2SO4, 160°C, 5 min) using three representative types of reactors: ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. After 96 h of enzymatic digestion, biomass treated in the SG and HS reactors achieved much higher cellulose conversions, 88% and 95%, respectively, compared to the conversion obtained using the ZC reactor (68%). Imaging at the micro- and nanoscales revealed that the superior performance of the SG and HS reactors could be explained by reduced particle size, cellular dislocation, increased surface roughness, delamination, and nanofibrillation generated within the biomass particles during pretreatment. CONCLUSIONS: Increased cellular dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of reactors that augment pretreatment with physical energy.
Project description:Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 ?m inner diameter (ID) × 160 mm, 180 ?m ID × 110 mm or 250 ?m ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a "fit-for-all" material. We also discuss issues related to the repeatability of monolith-preparations.
Project description:Microreactors present innovative solutions for problems pertaining to conventional reactors and therefore have seen successful application in several industrial processes. Yet, its application in heterogeneously catalyzed gas-liquid reactions has been challenging, mainly due to the lack of an easy and flexible methodology for catalyst incorporation inside these reactors. Herein, we report a facile technique for obtaining small (<2 nm) and well-distributed catalytic nanoparticles on the walls of silica-coated capillaries, that act as micro(channel) reactors. These particles are formed in situ on the reactor walls using polyelectrolyte multilayers (PEMs), built by layer-by-layer self-assembly. Manipulating the PEMs' synthesis condition gives easy control over metal loading, without compromising on particle size. Both monometallic (Au and Pd) and bimetallic (AuPd) nanoparticles were successfully obtained using this technique. Finally, these catalytic microreactors were found to exhibit exceptional activity for the direct synthesis of hydrogen peroxide from H<sub>2</sub> and O<sub>2</sub>.