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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycosylation is one of the most important posttranslational modifications affecting the functions of proteins and cell activities. Mass spectrometry (MS) has proven to be an effective tool for structural glycobiology and has helped gain an understanding of glycoprotein-mediated diseases. Although electro-spray ionization-tandem MS remains widely recognized as an effective means for oligosaccharide characterization, the hydrophilic nature of glycans has often caused the poor ionization efficiency requiring either derivatization or nanoelectrospray to improve detection sensitivity. In this report we describe the use of a chip-based infusion nanoelectrospray platform coupled with the hybrid triple quadrupole/linear ion trap for identification and characterization of glycosylation in complex mixtures. The high-mannose-type N-glycosylation in ribonuclease B was used to map the glycosylation site and obtain glycan structures. Using the chip-based nanoelectro-spray with precursor ion scanning linear ion trap MS, we were able to map the glycosylation site and obtain the glycan structures in ribonuclease B at 100 fmol/microL in a single analysis. In addition, a new, low-abundant glycoform with an additional hexose (Hex10GlcNAc2) attached to ribonuclease B was discovered. The results reported here demonstrate that the chip-based infusion nanoelectrospray ionization coupled to a quadrupole/linear ion trap platform is a valuable system, as it provides high sensitivity and stability for nanoelectrospray analysis, and allows extended acquisition time for completing precursor ion scanning and subsequent MS2 and MS3 information in a single analysis.
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PMID:Characterization of protein glycosylation using chip-based nanoelectrospray with precursor ion scanning quadrupole linear ion trap mass spectrometry. 1646 44

Microchip was coupled with MS through a stable, sensitive, and controllable sheath-flow nanoelectrospray (nES) interface for glycoprotein and glycopeptide analysis. The nano-ESI (nESI) was made with a delivery capillary, a commercial nES capillary, and a stainless steel (SS) tube which were connected together through a tee unit. High voltage for nES was applied on the SS tube and the commercial nES capillary was used as nES emitter. The delivery capillary was attached to the microchannel for delivering liquid from microchip to the nESI source. The flow rate of sheath liquid was optimized to be 100-200 nL/min which largely reduced the sample dilution. The detection limit of peptides on this microchip/MS platform was at femtomole level. Glycoprotein and glycopeptides were also successfully analyzed on the platform. All the glycoforms and glycopeptides of ribonuclease B (RNase B) were identified with this method. Some structures of the glycopeptides from RNase B were further characterized with MS/MS on the microchip, coupled with a quadrupole IT-MS.
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PMID:A sheath-flow nanoelectrospray interface of microchip electrophoresis MS for glycoprotein and glycopeptide analysis. 1711 89

The research on glycoproteomes represents an interesting field in the functional proteomics research. Affinity chromatography and mass spectrometry are powerful techniques that are used for gaining valuable information on glycoproteomes because glycoproteins and their unusual forms resulting from protein glycosylation can be important indicators of several diseases. In this study, the concanavalin A (Con A) immobilized silica packing was prepared and used for the separation of glycoprotein and glycopeptides. A very low, non-specific adsorption on the Con A affinity column was demonstrated by mass recovery of bovine serum albumin at more than 98.5%. The effect of concentration of methyl-alpha-D-mannopyranoside (alpha-Me-D-Man) in the mobile phase and the effect of flow rate on the retention behavior of ribonuclease B (RNase B) were also investigated. The standard glycoprotein RNase B was separated under optimized conditions using 0.2 mol/L alpha-Me-D-Man in the mobile phase at a flow rate of 0.5 mL/min. Meanwhile, the oligosaccharides and glycopeptides were enriched using a Con A column after digestion of the purified RNase B with peptide-N-glycosidase F (PNGase F) and trypsin. The structure of N-linked glycan and the rate and the site of glycosylation of RNase B were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Glycoproteins and glycopeptides in human serum and digest solution could be separated by this method. The results showed that this method is rapid and sensitive for the purification and characterization of glycoproteins and glycopeptides.
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PMID:[Preparation of a concanavalin A immobilized affinity column and its application in the structural analysis of ribonuclease B]. 1716 31

An automated analytical approach is proposed for simultaneous characterization of glycan and peptide moieties in pronase-generated glycopeptides. The proposed method is based on the use of a new pronase-immobilized enzyme reactor for the on-line rapid digestion of the target glycoprotein. By coupling the bioreactor to a Hypercarb chromatographic trap column, on-line selective glycopeptide enrichment prior to normal-phase liquid chromatography-mass spectrometry was obtained. A detailed study was carried out for integration and automation of each phase of the proposed analytical procedure. On-line digestion allowed extensive cleavage of the model protein (ribonuclease B), yielding to glycopeptides with peptide moieties up to eight amino acids, carrying the Man5-Man9 N-glycans each, selectively resolved on an Amide-80 column. The use of a linear ion trap instrument resulted in efficient ion capture and led to MS3 acquisition times and spectra quality similar to those for MS2, allowing the unambiguous identification of glycan (MS2) and peptide (MS3) sequences. The proposed procedure reduces the glycoprotein analysis time from approximately 3 days, as in most of the traditional off-line methods, to approximately 1 h.
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PMID:Pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MSn. 1719 61

A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved approximately 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.
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PMID:"One-pot" methylation in glycomics application: esterification of sialic acids and permanent charge construction. 1741 Oct 71

Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF(Fbs1) ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man3GlcNAc2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol.
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PMID:Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase. 1772 Jan 38

An improved method for site-specific characterization of protein glycosylation has been devised using nonspecific digestion with immobilized pronase combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This procedure was demonstrated using ribonuclease B (RNase B) and kappa-casein (kappa-csn) as representative N-linked and O-linked glycoproteins, respectively. Immobilization of the pronase enzymes facilitated their removal from the glycopeptide preparations, and was found to prevent enzyme autolysis while leaving the proteolytic activities of pronase intact. Increased digestion efficiency, simplified sample preparation, and reduced sample complexity were consequently realized. To supplement this technique, a refined glycopeptide search algorithm was developed to aid in the accurate mass based assignment of N-linked and O-linked glycopeptides derived from nonspecific proteolysis. Monitoring the progress of glycoprotein digestion over time allowed detailed tracking of successive amino acid cleavages about the sites of glycan attachment, and provided a more complete protein glycosylation profile than any single representative time point. This information was further complemented by tandem MS experiments with infrared multiphoton dissociation (IRMPD), allowing confirmation of glycopeptide composition. Overall, the combination of immobilized pronase digestion, time course sampling, FTICR-MS, and IRMPD was shown to furnish an efficient and robust approach for the rapid and sensitive profiling of protein glycosylation.
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PMID:Site determination of protein glycosylation based on digestion with immobilized nonspecific proteases and Fourier transform ion cyclotron resonance mass spectrometry. 1782 34

Detailed structural analysis of glycoproteins requires methods capable of isolating glycopeptides from tryptic digests of purified glycoproteins and complex protein mixtures. Here, we describe the selective and reproducible isolation of glycopeptides from a peptide mixture using ion-pairing normal-phase chromatography (IP-NPLC). The addition of inorganic monovalent ions in normal-phase chromatography appears to increase the hydrophobicity difference between peptides and glycopeptides, allowing for more efficient separation. Our data show that IP-NPLC effectively enriches glycopeptides from a tryptic digest of ribonuclease B, bovine fetuin, and a complex mixture of glycoproteins, when compared with normal-phase chromatography alone. The results of the IP-NPLC experiments can be explained using the Wimley-White water/octanol free energy scale to illustrate the hydrophobicity difference of nonglycosylated peptides with and without ion-pairing. We believe that IP-NPLC will be an important tool in glycoprotein characterization and glycoproteomic studies.
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PMID:Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography. 1797 48

A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins.
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PMID:Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity. 1802 44

Despite the increasing attention being paid to the functions of glycoproteins, their structural analysis is still difficult and hinders functional investigations. Structural analysis of post-translationally modified proteins is thought to be achieved using methods frequently utilized in proteomics research; however, the same methods cannot be used for glycosylated proteins. One of the difficulties associated with the physiochemical properties of glycopeptides and peptides is that the detection of the former is considerably more difficult, because of the existence of glycoforms that increase molecular weight and reduces quantities of individual species. Thus, difficulties are often faced in finding glycopeptide(s) by using MS when analyzing peaks (or fractions) obtained after proteolytic digestion and HPLC. One simple yet difficult solution to this problem would be to develop a purification method that provides better resolution. Our intention has been to address this issue by using a combination of conventional methods. We found that a method consisting of a combination of rough fractionation using a reverse-phase cartridge column under acidic conditions and comparative RP-HPLC, where the two chromatograms obtained using phosphate and borate buffers under basic conditions were compared, is effective for MS-based structural analysis. The applicability of the method in glycoprotein analysis was examined using various samples including ribonuclease B (RNase B), IgG1, ovalbumin (OVA), and asialo fetuin (ASF). The results suggest that the method is useful in the analysis of glycoproteins.
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PMID:Comparative RP-HPLC for rapid identification of glycopeptides and application in off-line LC-MALDI-MS analysis. 1817 86


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