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)

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Several phosphopeptide isolation strategies have been presented in the literature, including immobilized metal ion affinity chromatography. However, that technique suffers from poor selectivity and reproducibility. Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for phosphopeptide isolation prior to mass spectrometric analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two chromatographic materials possess complementary properties. For alpha- and beta-casein, Glu-C digestion provided no evident advantage compared to trypsin digestion when combined with TiO2 or ZrO2 phosphopeptide enrichment.
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PMID:Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. 1653 6

Proteolytic activity of polyclonal IgG antibodies (Abs) from the blood of AIDS patients was analyzed for the first time. These Abs were shown to display higher activity in hydrolysis of beta-casein than in hydrolysis of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT) or human serum albumin (HSA). Several abzymatic criteria were applied and it was shown that RT, HSA, and beta-casein hydrolyzing activities are an intrinsic property of polyclonal Abs from AIDS patients. Casein-hydrolyzing Abs were detected in the blood serum for 95% of AIDS patients, and it was shown that they possess serine protease-like catalytic activity. The substrate specificities of polyclonal Ab proteases and typical human proteases are different. Depending on the patient, the IgGs exhibit various pH optima of proteolytic activity. The products of casein hydrolysis by Ab proteases were different from those in the case of trypsin, chymotrypsin, and proteinase K.
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PMID:Proteolytic activity of IgG antibodies from blood of acquired immunodeficiency syndrome patients. 1654 61

In the present study, a human mammary epithelial cell (HMEC) culture model was developed to evaluate the potential involvement of carrier-mediated transport systems in drug transfer into milk. Trypsin-resistant HMECs were seeded on Matrigel-coated filters to develop monolayers of functionally differentiated HMEC. Expression of the specific function of HMEC monolayers was dependent of the number of trypsin treatments. Among the monolayers with different numbers of treatment (treated 1 to 3 times), the monolayer treated 3 times (3-t-HMEC monolayer) showed the highest maximal transepithelial resistance and expression of beta-casein mRNA as an index of differentiation. Transport of tetraethylammonium (TEA) across the 3-t-HMEC monolayer in the basolateral-to-apical direction was significantly higher than that in the apical-to-basolateral direction (p < 0.05), whereas such directionality was not observed for p-amino-hippurate, suggesting the existence of organic cation transporters, but not organic anion transporters. In fact, expression of mRNAs of human organic cation transporter (OCT) 1 and 3 were detected in the 3-t-HMEC monolayer. These results indicate that the 3-t-HMEC monolayer is potentially useful for the evaluation of carrier-mediated secretion of drugs including organic cations into human milk.
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PMID:Development of a human mammary epithelial cell culture model for evaluation of drug transfer into milk. 1675 89

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 microm, o.d. 360 microm) 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 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified 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 1fmol/mul (1nM) 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.
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PMID:Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping. 1707 19

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is approximately 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipetting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.
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PMID:Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes. 1726 46

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from beta-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using beta-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.
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PMID:Protein digestion and phosphopeptide enrichment on a glass microchip. 1772 69

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.
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PMID:Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins. 1825 78

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).
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PMID:Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions. 1834 89

An enzyme-immobilized capillary microreactor for rapid protein digestion and proteomics analysis is reported. The inner surface of the fused-silica capillary was coated with poly(diallyldimethylammonium chloride) (PDDA)-entrapped silica sol-gel matrix, followed by assembly of trypsin onto the PDDA-modified surface via electrostatic adsorption. The immobilization parameters such as PDDA content in the sol-gel matrix, trypsin concentration and pH were investigated in detail. Protein samples including beta-casein, myoglobin and cytochrome c could be effectively digested and electrophoretically separated simultaneously in such a modified capillary. Just 2.26 ng (corresponding to 0.10-0.14 picomole) of sample was sufficient for on-line capillary electrophoresis peptide mapping. The efficiency of the digestion was further demonstrated by digestion of a human liver cytoplasm sample and 253 proteins were identified in one unique run.
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PMID:Trypsin entrapped in poly(diallyldimethylammonium chloride) silica sol-gel microreactor coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1838 13

The catalytic performance of porous silicon (PS) micro enzyme reactors (muIMER) is strongly dependent on the PS matrix morphology for enzyme immobilisation. PS was achieved in the muIMER by anodisation in a HF-ethanol mixture. PS etching of structured silicon surfaces commonly results in an inhomogeneous pore formation. The deep channel microreactors described herein have previously suffered from these phenomena, yielding non-optimised muIMERs. In order to obtain a homogeneous PS layer on the deep microreactor channel walls, different reactor geometries (channel wall thicknesses of 50 and 75 mum) were anodised at 10 and 50 mA cm(-2) for anodisation times ranging between 0 and 50 min. The muIMERs were evaluated by immobilising two types of enzymes, glucose oxidase (GOx) and trypsin, and the resulting catalytic turnover was monitored by a colorimetric assay. It was found that reactors with a homogeneous PS matrix displayed improved performance. The trypsin muIMERs were used to digest a protein, beta-casein, in an on-line format and the digest was analysed by MALDI-TOF MS. The importance of tailoring the muIMER geometry and the PS-matrix is crucial for the protein digestion. Successful protein identification after only 12 s. digestion was demonstrated for the best reactor, 75 mum channel wall, 25 mum channel width, anodised at 50 mA cm(-2) for 10 min.
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PMID:Improved performance in silicon enzyme microreactors obtained by homogeneous porous silicon carrier matrix. 1896 6


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