Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor (original) (raw)
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Enzyme-immobilized reactors for rapid and efficient sample preparation in MS-based proteomic studies
PROTEOMICS, 2013
Proteolysis is a key step in proteomic studies integrated with MS analysis but the conventional method of in-solution digestion is limited by time-consuming procedures and low sensitivity. Furthermore, obtaining reliable peptide maps and meaningful sequence data using MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions. Recently, various immobilized-enzyme reactors have been developed for highly efficient proteolysis in MS-based proteomic analysis. This review focuses on the proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. We describe the preparation of enzyme reactors by several techniques and protein digestion under unusual conditions. Analysis of posttranslational modifications by enzyme reactors prepared using our immobilization method is presented as a model application. Analysis systems using immobilized-enzyme reactors are expected to become useful tools for proteomic studies and diverse applications in biotechnology.
Journal of Proteome Research, 2006
A nanoliter trypsin-based monolithic microreactor coupled with µRPLC-MS/MS was reported for shotgun proteome analysis. The proteins were rapidly digested by the microreactor, and the resulting protein digests were directly loaded onto a µRPLC column for separation followed with detection of the eluted peptides by tandem mass spectrometer. The digestion efficiency and stability of the microreactor was demonstrated by using bovine serum albumin as a model protein. When compared with an incubation time of more than 10 h by free trypsin in the conventional digestion approach, protein mixtures can be digested by the microreactor in several minutes. This system was applied to the analysis of the total cell lysate of Saccharomyces cerevisiae. After a Sequest database search, a total of 1578 unique peptides corresponding to 541 proteins were identified when 590 ng yeast protein was digested by the microreactor with an incubation time of only 1 min.
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.
Journal of Proteome Research, 2008
A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5-35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional "overnight" sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.
2005
The applicability of a trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS for rapid proteolytic digestion and protein identification is here described. Dilute samples are passed through the bioreactor for generation of proteolytic fragments in less than 10 min. After digestion and peptide separation, electrospray ionization tandem mass spectrometry is used to generate a peptide map and to identify proteolytic peptides by correlating their fragmentation spectra with amino acid sequences from a protein database. By digesting picomoles of proteins sufficient data from ESI and MS/MS were obtained to unambiguously identify proteins alone and in serum samples. This approach was also extended to locate mutation sites in beta-lactoglobulin A and B variants.
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.