Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase.

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine(1----4)]n (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.     

κ-卡拉胶寡糖的反相离子对-超高效液相色谱-四极杆-飞行时间质谱研究

建立反相离子对-超高效液相色谱(RPIP-UPLC)和电喷雾离子源-四极杆-飞行时间质谱(ESI-Q-TOF-MS)联用技术快速分离鉴定硫酸寡糖的方法.以20 mmol/L庚胺(pH 4)为离子对试剂,25%庚胺甲酸盐纯水溶液(A)和25%庚胺甲酸盐甲醇溶液(B)为梯度洗脱溶剂,κ-卡拉胶寡糖通过BEH C18反相柱分离后,分别在正、负离子模式下进行四极杆-飞行时间质谱分析.结果表明,聚合度为3～45的κ-卡拉胶寡糖在BEH C18柱上得到很好的分离,从每一个色谱峰对应的质谱图中可以准确获得直至27糖的各寡糖结构信息,均为奇数糖,与聚丙烯凝胶电泳结果吻合.所得的寡糖断裂规律对卡拉胶寡糖的快速鉴定和结构解析具有重要意义.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma. Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999     

Electroblotting of Immobiline DryPlates applied to identification of human plasma apolipoprotein A-I

A method for efficient electroblotting of Immobiline DryPlates, allowing subsequent immunological identification of separated proteins has been developed. A thin layer of 1% agarose containing sodium dodecyl sulfate is moulded on the 0.5 mm thick polyacrylamide gel surface after completed electrophoresis. After separation of the agarose-polyacrylamide gel sandwich from the plastic film the rigid gel sandwich could be easily transferred to a nitrocellulose membrane and electroblotting could be performed without adherence of the sticky polyacrylamide gel layer to the membrane. Using this technique human plasma high density apolipoprotein A-I isoforms, over a wide concentration range, could be identified in a heterogeneous mixture, conserving the isoform pattern and band sharpness produced in the immobilized pH gradient experiments.     

Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid: application to the enzymological structural analysis of oligosaccharides

Mono- and oligosaccharides, each containing a reducing end group, were labeled with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid and the resulting derivatives were separated with high resolution by polyacrylamide gel electrophoresis using a method developed recently (P. Jackson, Biochem. J. 1990, 270, 705-713) but with an alternative electrophoretic buffer system. The fluorescent derivatives of glucose and all its straight chain, alpha 1-4 linked, oligomers from maltose to maltoheptaose were well resolved. Various isomers such as maltose and lactose could be separated, as were maltose and cellobiose and some epimers, for instance glucose and galactose. The method was applied to the analysis of the partial sequential degradation of a complex oligosaccharide with neuraminidase and beta-galactosidase. Gels showing fluorescent saccharide band patterns were recorded in picomolar quantities either photographically or using an imaging system based on a cooled charge-coupled device.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry

Oligosaccharides were derivatized by reductive amination using benzylamine and analyzed by nanoelectrospray ionization-quadrupole time of flight-tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+H]+ ions for all benzylamine-derivatized oligosaccharides. To obtain structural information from these derivatized oligosaccharides, MS/MS was applied. Protonated molecular ions underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M+H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. Fragmentation of the [M+H]+ ions gave predominantly B- and Y-type glycosidic fragments. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could easily be obtained. Predictable and reproducible fragmentation patterns could be obtained in all cases. This derivatization procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from 10 microg of glycoproteins separated by gel electrophoresis. Moreover, the derivatives retain their sensitivity to exoglycosidases. Thus a series of sequential on-target exoglycosidase treatments combined with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was found to be useful for the determination of structural features of the glycans released from proteins separated by gel electrophoresis such as the monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. Our strategy can be used successfully to assign the major glycans released from proteins separated by gel electrophoresis.     

Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis

Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.     

Polyacrylamide gradient gel electrophoresis of cytosolic retinol- and retinoic acid-binding proteins: application to rat testis and liver

Distribution and cellular levels of retinol-binding protein and retinoic acid-binding protein, involved in the molecular action of retinoids, were analyzed in rat testis and liver. Both binding proteins of cytosolic extracts were separated by linear-polyacrylamide gradient gel electrophoresis and following electrophoretic separation, could be visualized by complementary identification tests such as autoradiography and marker proteins. The concentration of the binding proteins were evaluated by scanning the polyacrylamide gradient gels and the resulting data were found to be in accordance with those obtained by counting radioactivities. Polyacrylamide gradient gel electrophoresis appears suitable to detect and quantitatively evaluate cytosolic retinol- and retinoic acid-binding proteins.     

系列ι-卡拉胶寡糖制备及其电喷雾串联质谱序列分析

以ι-卡拉胶为原料,在稀酸条件下进行酸水解得到其寡糖混合物,采用低压凝胶渗透色谱(LPGPC)进行分离纯化,获得了10个寡糖单体.在利用聚丙烯酰胺凝胶电泳(PAGE)和高效薄层层析(HPTLC)对其纯度进行分析的基础上,通过红外光谱(IR)、核磁共振波谱(NMR)和电喷雾离子化质谱(ESI-MS)对其结构进行表征,并用电喷雾碰撞诱导串联质谱(ESI-CID-MS/MS)对其序列进行分析.结果表明,它们分别是还原端为2-硫酸-3,6-内醚半乳糖(A2S)和非还原端为4-硫酸-半乳糖(G4S)的ι-卡拉胶二至二十糖.这些酸法水解制备的寡糖结构新颖,不同于ι-卡拉胶酶法制备的新ι-卡拉胶寡糖.这不仅丰富了海洋寡糖库数据,也为进一步运用糖生物芯片技术探索其与蛋白之间的相互作用提供了物质基础.     

N-linked oligosaccharide analysis of rat brain Thy-1 by liquid chromatography with graphitized carbon column/ion trap-Fourier transform ion cyclotron resonance mass spectrometry in positive and negative ion modes

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ?M - H(-) ions.     

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract

The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.     

Monitoring the N-glycosylation of plant glycoproteins by fluorophore-assisted carbohydrate electrophoresis

We have evaluated the efficiency of a fast, simple and efficient method, fluorophore-assisted carbohydrate electrophoresis (FACE), for the characterization of plant N-linked glycans. After their enzymatic release from plant glycoproteins, N-glycans were reductively aminated to the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. In addition, an affinity purification procedure using concanavalin A was developed for separation of ANTS-labeled high-mannose-type N-glycans from other plant oligosaccharides.     

Gel electrophoretic analysis of chitosan hydrolysis products.

Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5% v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R-250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10% w/v polyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33% w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R-250 was found to be better than Calcofluor White M2R for staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system. To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.     

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.     

Analysis of histones by on-line capillary zone electrophoresis-electrospray ionisation mass spectrometry

Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.     

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.     

Important parameters in semi-dry electrophoretic transfer.

The efficiency of semi-dry electrophoretic transfer after sodium dodecyl sulfate (SDS)-electrophoresis using PhastGel media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.