Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.
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PMID:Mass spectrometry of protein-ligand complexes: enhanced gas-phase stability of ribonuclease-nucleotide complexes. 1856 58

Recombinant proteins have been previously synthesized in a transgenic rice cell suspension culture system with the rice amylase 3D promoter, which can be induced via sugar starvation. However, the secreted recombinant proteins have been shown to be rapidly decreased as the result of proteolytic degradation occurring during prolonged incubation. The secreted proteases were identified via two-dimensional electrophoresis (2-DE) and ESI/Q-TOF mass spectrometry analyses. The internal amino acid sequences of 8 of 37 spots corresponded to cysteine proteinase (CysP), which is encoded for by Rep1 and EP3A. This result shows that CysP is a major secreted protease in rice cell suspension cultures following induction via sugar starvation. Intron-containing self-complementary hairpin RNA (ihpRNA)-mediated post-transcriptional gene silencing (PTGS) was applied to suppress the expression of CysP in rice cell suspension cultures. The reduction of rice CysP mRNA and the detection of siRNA specific to CysP, an initiator of RNAi, were verified via Northern blot analysis and RNase protection assays, respectively, thereby indicating that PTGS operated successfully in this system. The analysis of total secreted protease and CysP activities evidenced lower activity than was observed with the wild-type. Furthermore, suspension cultures of rice cells transformed with both hGM-CSF and the gene expressing the ihpRNA of CysP evidenced a reduction in total protease and CysP activities, and an up to 1.9-fold improvement in hGM-CSF production as compared to that observed in a rice cell line expressing hGM-CSF only. These results demonstrate the feasibility of the suppression of CysP via RNA interference to reduce protease activity and to increase target protein accumulation in rice cell suspension cultures.
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PMID:Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. 1858 53

We developed a mass spectrometric method to determine the p K a values of individual histidine residues in proteins. The method is based on the fact that the imidazole C 2-proton undergoes pH-dependent hydrogen-deuterium exchange reaction, of which the rate constant ( k phi) reflects the p K a for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D 2O solvent at various pH values, (2) protein digestion by proteolytic enzyme(s), during which all the rapidly exchanging deuterons such as those in amide and hydroxyl groups are back-exchanged for protons, and (3) measurement of the mass spectrum of each histidine-containing peptide by LC/ESI-MS. The k phi of the H-D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The p K a value is then determined from a plot of k phi versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the p K a values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The p K a values of three of four histidine residues (His12, -105, and -119) in RNase A were successfully determined by this method and were in good agreement with those determined by (1)H NMR and hydrogen-tritium exchange methods. The method uses subnanomole quantities of protein, allowing measurement at a much lower concentration than that of 1 mM required for the conventional NMR approach that is currently almost exclusively the method of choice.
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PMID:Determination of pKa values of individual histidine residues in proteins using mass spectrometry. 1866 14

Two approaches for the evaluation of the relative degree of global DNA methylation through the quantification of 2' deoxynucleosides are described. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA is performed using both high-performance capillary electrophoresis (HPCE) with UV-Vis detection or liquid chromatography with electrospray ionization mass spectrometric detection (LC-ESI/MS). Treatment of genomic DNA with a ribonuclease and generation of nucleosides through enzymatic hydrolysis notably increases the specificity of both techniques. Both approaches have been demonstrated to be highly specific and sensitive, being useful for the rapid quantification of the degree of global DNA methylation and its exploitation for the analysis of poorly purified and/or concentrated DNA samples, such as tumor biopsies.
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PMID:Quantification of global DNA methylation by capillary electrophoresis and mass spectrometry. 1898 3

Rapid, selective and sensitive determination of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N-linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N-linked oligosaccharides in various glycoproteins seems to be possible.
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PMID:Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry. 1910 23

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.
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PMID:Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans. 1941 47

The lipid oxidation product 4-oxo-2-nonenal (ONE) derived from peroxidation of polyunsaturated fatty acids is a highly reactive protein cross-linking reagent. The major family of cross-links reflects conjugate addition of side chain nucleophiles such as sulfhydryl or imidazole groups to the C triple bond C of ONE to give either a 2- or 3-substituted 4-ketoaldehyde, which then undergoes Paal-Knorr condensation with the primary amine of protein lysine side chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or beta-alanylhistidine (carnosine), this would lead to circulating 4-ketoaldehydes that could then bind covalently to the protein Lys residues. This phenomenon was investigated by SDS-PAGE and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and LC-ESI-MS/MS with both tryptic and chymotryptic digestion). Under the reaction conditions of 0.25-2 mM ONE, 1 mM GSH or carnosine, 0.25 mM bovine beta-lactoglobulin (beta-LG), and 100 mM phosphate buffer (pH 7.4, 10% ethanol) for 24 h at 37 degrees C, virtually every Lys of beta-LG was found to be fractionally cross-linked to GSH. Cross-linking of Lys to carnosine was less efficient. Using cytochrome c and RNase A, we showed that ONE becomes more protein-reactive in the presence of GSH, whereas protein modification by 4-hydroxy-2-nonenal is inhibited by GSH. Stable antielectrophile-ONE-protein cross-links may serve as biomarkers of oxidative stress and may represent a novel mechanism of irreversible protein glutathionylation.
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PMID:Covalent cross-linking of glutathione and carnosine to proteins by 4-oxo-2-nonenal. 1948 Mar 92

This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contain four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side-chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5 % acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side-chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.
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PMID:Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages. 1995 38

Procainamide was investigated as a multifunctional oligosaccharide label for glycan profiling and identification in a HPLC-FL/ESI-QTOF system. Addition of this aromatic amine to glycans through reductive amination improves fluorescence detection and ESI ionization efficiency. Both procainamide and 2-AB derivatives of N-linked glycans released from three glycoproteins (Human IgG, Mouse IgG, and RNase B) were quantitatively profiled with HPLC-FL and identified with ESI-QTOF. The procainamide derivatives produced FL glycan profiles comparable to the 2-AB derivatives, but with a few extra minor peaks, which suggests better labeling efficiency for procainamide derivatives for minor peaks. The procainamide derivatives also improve ESI ionization efficiency by 10-50 times over the respective 2-AB derivatives and the ESI-QTOF method sensitivity is at the low picomole to high femtomole level. Using the procainamide tag, all N-linked glycans released from three tested glycoproteins can be quantitatively detected with HPLC-FL and identified with ESI-QTOF at the same time. Monosaccharide sequence confirmation was also demonstrated in this study.
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PMID:The evaluation of a novel approach for the profiling and identification of N-linked glycan with a procainamide tag by HPLC with fluorescent and mass spectrometric detection. 2041 45

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC-HILIC column and ESI-MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC-HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.
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PMID:Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection. 2088 7


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