Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification (original) (raw)
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Development of an open-tubular trypsin reactor for on-line digestion of proteins
Analytical and Bioanalytical Chemistry, 2007
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°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 μL −1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm 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.
Analytical Biochemistry, 2006
A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 m, o.d. 360 m) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1 pmol/ l angiotensin II, cytochrome c, hemoglobin, and -casein. Because the inner walls of the capillaries are modiWed by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1 fmol/ l (1 nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.
Multi-lumen capillary based trypsin micro-reactor for the rapid digestion of proteins
The Analyst, 2018
IIn this work we evaluated a novel microreactor prepared using a surface modified, high surface-tovolume ratio multi-lumen fused silica capillary (MLC). The MLC investigated contained 126 parallel channels, each of 4 µm internal diameter. The MLC, along with conventional fused silica capillaries of 25 µm and 50 µm internal diameter, were treated by (3-aminopropyl)triethoxysilane (APTES) and then modified with gold nanoparticles, of ∼20 nm in diameter, to ultimately provide immobilisation sites for the proteolytic enzyme, trypsin. The modified capillaries and MLCs were characterised and profiled using non-invasive scanning capacitively coupled contactless conductivity detection (sC4D). The sC4D profiles confirmed a significantly higher amount of enzyme was immobilised to the MLC when compared to the fused silica capillaries, attributable to the increased surface to volume ratio. The MLC was used for dynamic protein digestion, where peptide fragments were collected and subjected to off-line chromatographic evaluation. The digestion was achieved with the MLC reactor, using a residence time of just 1.26 min, following which the HPLC peak associated with the intact protein decreased by >70%. The MLC reactors behaved similarly to the classical in vitro or in-solution approach, but provided a reduction in digestion time, and fewer peaks associated with trypsin auto-digestion, which is common using in-solution digestion. The digestion of cytochrome C using both the MLC-IMER and the in-solution approach, resulted in a sequence coverage of ∼80%. The preparation of the MLC microreactor was reproducible with <2.5% RSD between reactors.
Trypsin immobilization on three monolithic disks for on-line protein digestion
Journal of Pharmaceutical and Biomedical Analysis, 2008
The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carbonyldiimidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media ® (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280 nm, was comprised between 0.9 and 1.5 mg per disk. Apparent kinetic parameters K * m and V * max , as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-␣-benzoyl-l-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, ␣ 1 -acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI-MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM ® EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5 min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12 M) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.
Analytica Chimica Acta, 2010
The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca 2+ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the IMER were investigated for the substrate -casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of -casein could be achieved in a few minutes (86 s residence time in the IMER followed by a wash step).
Enzymatic protein digest in chip-based nanovials with immobilized proteolytic enzymes
Analytica Chimica Acta, 2005
In the present work, protein digest reactions in silicon-based microchips, coated with immobilized proteolytic enzymes, have been carried out. The performance of such vials, modified with trypsin or chymotrypsin, was tested with myoglobin as a substrate. Capillary electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry were utilized for analysis of the digests, and the influence of different instrumentation setups, immobilization procedures and reaction conditions are discussed.
Scientific Reports, 2013
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 mm 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 mm 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 mm 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.
Integrated Microanalytical Technology Enabling Rapid and Automated Protein Identification
Analytical Chemistry, 2000
Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (μ-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The μ-chip IMER provides on-line enzymatic digestion of protein samples (1 μL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).
Journal of Chromatography A, 2009
The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The microimmobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.
Sensors and Actuators B: Chemical, 2009
We have developed, fabricated and tested a lidless polymeric microchip which integrates a microreactor with an electrospray ionization (ESI) tip for mass spectrometric (MS) analysis. The microchip contains a microreactor spot, a micropillar-filled channel and an electrospray tip, all made of an SU-8 epoxy polymer. The fabrication process of the microchips required just two lithographic steps, making it simple, cost-effective, rapid, and well-suited for mass production. The lidless structure of the chip provides easy sampling of reagents and samples. On-chip trypsin protein digestion of bovine heart cytochrome c (CytC) and bovine serum albumin (BSA) was performed on the microreactor in a solvent drop without the need for immobilization of the enzyme to the microreactor. The digestion was accomplished in the microreactor within 8 min, after which the digestion products were measured directly by an integrated on-chip ESI combined to MS. The whole procedure was rapid as the total time for on-chip digestion and measurement was less than 10 min. An average sequence coverage obtained with CytC was 90% and with BSA it was 82%. The microchip can also be used as a stand-alone electrospray ionization tip for mass spectrometric analysis.