Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.
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PMID:Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator. 864 33

Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha2-HS glycoprotein (human fetuin) and alpha1-antitrypsin (alpha1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE.
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PMID:Analyzing glycoproteins separated by two-dimensional gel electrophoresis. 963 44

The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. N-Glycosylation is needed for proper membrane insertion, EGF binding and receptor functioning. The human epidermoid carcinoma A431 cell line secretes a soluble 105 kDa glycoprotein (sEGFR) that represents the extracellular domain of the membrane-bound form, and its glycosylation pattern has been investigated. After liberation of the oligosaccharides from sEGFR with PNGase F, the glycans were fractionated along different routes, including Concanavalin A affinity chromatography, anion-exchange chromatography, HPLC and high-pH anion-exchange chromatography. The oligosaccharide fractions were characterized by 500- and 600-MHz 1H-NMR spectroscopy and mass spectrometry (FAB, ESI, and MALDI-TOF). The oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2 and account for 17% of the total carbohydrate moiety. Furthermore, di-, tri'- and tetraantennary complex-type structures are present, both neutral and (alpha2-3)-sialylated (up to tetrasialo), comprising 24 and 59%, respectively, of the total carbohydrate moiety. In this study, 32 new complex-type glycans are characterized containing the Le(x), Le(Y), and sialyl-Le(x) determinants, the bloodgroup A and H antigens, as well as the ALe(Y) determinant. This first comprehensive glycosylation study on a human nonrecombinant receptor shows the immense heterogeneity of the glycosylation of sEGFR.
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PMID:Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor. 1098 52

The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and alpha-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc2Man5 approximately 9, and that attached to TSL has a structure of GlcNAc2Min5 approximately 8. The glycoform distributions in these glycoproteins are quite different, with the GlcNAc2Man5 type predominant in RNase B, and the GlcNAc2Man8 type, in TSL This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.
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PMID:Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion. 1179 56

A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.
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PMID:Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins. 1264 20

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.
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PMID:Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry. 1465 30

Human plasma-derived antithrombin was characterized in both the native and de-N-glycosylated forms (without separation of isoforms) by means of electrospray ionization ion trap mass spectrometry (ESI-ITMS). In order to determine the limits of the instrument set-up, the molecular mass precision and accuracy of the ESI-ITMS analysis was evaluated with the standard protein enolase and some instrumental data acquisition parameters were optimized. Mass precision was determined as a function of the number of averaged mass spectra (= scans) and data acquisition time. For this study, 20 and 50 scans were averaged and the data acquisition time was chosen to be between 0.5 and 5 min. It turned out that data acquisition times longer than approximately 2 min show no significant differences of the standard deviation of the determined molecular mass. Furthermore, the ion trap scan rate was varied at constant acquisition time of 2 min and the number of averaged scans was set to 20. At the scan rate of 13,000 u s(-1) a mass precision of +/-1.8 Da and a mass accuracy of +0.026% were determined. On reducing the scan rate to 5500 u s(-1), better agreement with the theoretical molecular mass was obtained, showing a mass accuracy of +0.012% but with a decrease in the mass precision to +/-3.0 Da. Using the optimized scan rate of 13,000 u s(-1) and a data acquisition time of 2 min, the exact molecular mass was determined of the three forms of antithrombin, namely the alpha-form, the beta-form and the natural mixture (present in human plasma) containing both forms. The protonated molecular masses were found to be 57,854 and 55,664 Da for the affinity chromatography-isolated alpha-and beta-form, respectively. The mass difference of 2190 Da is attributed to the known difference in carbohydrate content at one specific site. The protonated molecular mass of the dominating species of the natural mixture in human plasma was shown to be 57,850 Da, corresponding to the alpha-form, the major component in native plasma. In this mixture the beta-form was also detected, exhibiting a protonated molecular mass of 55,655 Da, but showing a much lower abundance, as expected. To obtain a complete release of the N-glycan residues by means of PNGase F, a denaturation, reduction and alkylation step of the glycoproteins was performed before the enzymatic reaction. After enzymatic removal of all N-glycans, the protonated molecular masses obtained were 49,399, 49,380 and 49,391 Da for the alpha-form, the beta-form and the unseparated natural mixture, respectively. These values are in good agreement (+0.026% for the alpha-form, -0.012% for the beta-form and +0.010% for the unseparated mixture) with the calculated molecular mass based on the SwissProt data. The determined molecular masses after reduction/alkylation and de-N-glycosylation of the alpha-and beta-forms are almost equal, indicating that no major differences exist between the three preparations on the amino acid level.
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PMID:Exact molecular mass determination of various forms of native and de-N-glycosylated human plasma-derived antithrombin by means of electrospray ionization ion trap mass spectrometry. 1557 42

This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.
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PMID:Use of multidimensional lectin affinity chromatography in differential glycoproteomics. 1585 96

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.
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PMID:Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase. 1644 50

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.
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PMID:N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis. 1724 9


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