Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, we demonstrated that microwave-assisted enzymatic digestion could be greatly accelerated by multifunctional magnetite beads. The acceleration of microwave-assisted enzymatic digestion by the presence of the magnetite beads was attributable to several features of the beads. Their capacity to absorb microwave radiation leads to rapid heating of the beads. Furthermore, their negatively charged functionalities cause adsorption of proteins with opposite charges onto their surfaces by electrostatic interactions, leading to a concentration on the surfaces of the beads of proteins present in trace amounts in the solution. The adsorbed proteins are denatured and hence rendered vulnerable to enzymatic digestion and are digested on the beads. For microwave heating, 30 s was sufficient for carrying out the tryptic digestion of cytochrome c, in the presence of magnetite beads, while 1 min was adequate for tryptic digestion of myoglobin. The digestion products were characterized by MALDI-MS. This rapid enzymatic digestion allowed the entire time for identification of proteins to be greatly reduced. Furthermore, specific proteins present in trace quantities were enriched from the sample on the magnetite beads and could be rapidly isolated from the sample by employing an external magnetic field. These multiple roles of magnetite beads, as the absorber for microwave irradiation, the concentrating probe, and the agent for unfolding proteins, contributed to their capability of accelerating microwave-assisted enzymatic digestion. We also demonstrated that trypsin immobilized magnetite beads were suitable for use in microwave-assisted enzymatic digestion.
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PMID:Acceleration of microwave-assisted enzymatic digestion reactions by magnetite beads. 1728 12

Characterisation and identification of peptides (800 to 5000 Da) generated by intestinal digestion of fish or meat were performed using MS analyses (matrix-assisted laser desorption ionisation time of flight and nano-liquid chromatography electrospray-ionisation ion trap MS/MS). Four pigs fitted with cannulas at the duodenum and jejunum received a meal exclusively made of cooked Pectoralis profundus beef meat or cooked trout fillets. A protein-free meal, made of free amino acids, starch and fat, was used to identify peptides of endogenous origin. Peptides reproducibly detected in digesta (i.e. from at least three pigs) were evidenced predominantly in the first 3 h after the meal. In the duodenum, most of the fish- and meat-derived peptides were characteristic of a peptic digestion. In the jejunum, the majority of peptides appeared to result from digestion by chymotrypsin and trypsin. Despite slight differences in gastric emptying kinetics and overall peptide production, possibly in relation to food structure and texture, six and four similar peptides were released after ingestion of fish or meat in the duodenum and jejunum. A total of twenty-six different peptides were identified in digesta. All were fragments of major structural (actin, myosin) or sarcoplasmic (creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and myoglobin) muscle proteins. Peptides were short ( < 2000 Da) and particularly rich in proline residues. Nineteen of them contained bioactive sequences corresponding mainly to an antihypertensive activity. The present work showed that after fish or meat ingestion, among the wide variety of peptides produced by enzymic digestion, some of them can be reproducibly observed in intestinal digesta.
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PMID:Peptides reproducibly released by in vivo digestion of beef meat and trout flesh in pigs. 1776 98

In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.
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PMID:On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis. 1785 14

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.
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PMID:Development of an open-tubular trypsin reactor for on-line digestion of proteins. 1789 35

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.
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PMID:A nanoporous reactor for efficient proteolysis. 1796 May 51

Because of enhanced a(1) ion signals, dimethyl labeling is useful for identifying the N-termini of proteins or peptides. Herein, we describe a novel strategy that uses dimethyl isotope-coded affinity selection (DICAS) to isolate peptides that contain either the dimethylated or in vivo blocked N-termini of proteins for comprehensive sequence analyses by LC-MS/MS. In this method, dimethyl labeling at the protein level was first performed using formaldehyde-d(2) to label all unblocked protein N-termini and lysine residues, followed by trypsin digestion. The free N-terminal amines of internal peptides generated by digestion were captured by solid supports with aldehyde functionalities through reductive amination. The flow-through fractions, which contained either in vivo or in vitro blocked N-terminal peptides, were subjected to sequence analyses by LC-MS/MS. Owing to the unique feature of a1 signal enhancement associated with dimethylated peptides and the use of the deuterium reagent, the in vitro blocked (or in vivo free) N-termini of proteins could be easily differentiated from the in vivo blocked N-termini. Thus, their sequences and N-terminal modifications could be assigned unambiguously from MS/MS spectra. In this study, the completeness of the labeling and the efficiency of the isolation method were first confirmed by the use of a mixture of model proteins composed of hemoglobin, myoglobin, and alpha-lactalbumin. The N-termini of all three proteins, including both alpha and beta chains of hemoglobin as well as a signal sequence located in the N-termini of alpha-lactalbumin, were successfully identified. The protocol was then applied to the analysis of an unfractionated lysate of MCF-7 cells. Results indicate that more than 80% of the isolated peptides contained the N-termini of unique proteins, and many of them were consistent with known or inferred N-terminal processing such as methionine removal and acetylation. In addition, using the DICAS approach, we identified a novel N-terminal formylation for the Ig kappa chain V-III region SIE protein and a novel N-terminal signal sequence (1th-32th amino acid) for profilin.
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PMID:Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC-MS/MS. 1800 Nov 27

An easily replaceable microchip enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized superparamagnetic nanoparticles. Magnetic nanoparticles with small size (50 nm in diameter) and strong magnetism were synthesized. At first, amine-functionalized magnetic nanoparticles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde (-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The prepared magnetic nanoparticles were then locally packed onto the glass microchip by the application of a strong magnetic field using a magnet to form an on-chip magnetic nanoparticles packing bed. Capability of the proteolytic microreactor was demonstrated by cytochrome c, bovine serum albumin and myoglobin as model proteins. The digestion products were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with sequence coverage of 83%, 43% and 79% observed, respectively. Complete protein digestion was achieved in a short time (10 s) under the flow rate of 5 microL/min. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. It is easy to replace the nanoparticles and make the new microreactor. It takes less than 1 min under the condition of extra magnetic to form a new packing bed. The packing bed can be used for at least five times without any treatments. Additionally, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. After a database search, six proteins were identified. This opens a route for its further application in bottom-up proteomic analysis.
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PMID:On-chip enzymatic microreactor using trypsin-immobilized superparamagnetic nanoparticles for highly efficient proteolysis. 1802 85

Sequence-specific proteolysis is an important part of protein identification by MS. Digestion of protein is commonly performed in-solution, in sample vials with volumes ranging from milli- to microliters. When digestion is performed with a sample volume below 1 microL, handling of solution and potential sample loss via adsorption become significant issues. In this report, a proof of concept for the digestion of a small volume protein solution inside a capillary was demonstrated using a discontinuous buffer system previously studied (Nesbitt, C. A., et al. J. Chromatogr. A 2005, 1073, 175-180). Upon voltage application, a pH junction was created by the discontinuous buffer. Using myoglobin as an example, the protein molecules were enriched at the junction with an estimated volume of a few nanoliters. A protease, trypsin, was then introduced to myoglobin at the junction by coenrichment to induce in-capillary digestion. The voltage application was then suspended to provide the necessary time (2 h) for the proteolysis to proceed. When completed, voltage application was resumed, and the discontinuous buffer reconcentrated the peptides formed from digestion. Importantly, the refocused peptides appeared to roughly elute according to their pIs, resulting in a partial separation. Direct sample deposition from capillary was performed to facilitate mass spectral analysis by MALDI. The partial separation, according to pI, offered the potential benefits of MALDI MS signal enhancement and provided supplementary pI information for peptide identity assignment.
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PMID:Nanoliter-volume protein enrichment, tryptic digestion, and partial separation based on isoelectric points by CE for MALDI mass spectral analysis. 1808 Dec 1

In this work, a piece of glass fiber was inserted into the channel of a poly(methyl methacrylate) (PMMA) electrophoresis microchip to enhance the electroosmotic flow (EOF) and the separation efficiency. The EOF value of the glass fiber-containing microchannel at pH 8.2 was determined to be 4.17 x 10(-4)cm2 V(-1)s(-1). The performance of the new microchip was demonstrated by its ability to separate and detect three purines coupled with end-column amperometric detection. In addition, a piece of trypsin-immobilized glass fiber was inserted into the channel of a PMMA microchip to fabricate a core-changeable microfluidic bioreactor that can be regenerated by changing the fiber. The in-channel fiber bioreactor has been coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the digestion and peptide mapping of bovine serum albumin and myoglobin.
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PMID:Fabrication and performance of poly(methyl methacrylate) microfluidic chips with fiber cores. 1809 73

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 280nm, was comprised between 0.9 and 1.5mg per disk. Apparent kinetic parameters Km* and Vmax*, as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, alpha1-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 5min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12microM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.
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PMID:Trypsin immobilization on three monolithic disks for on-line protein digestion. 1824 15


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