Analysis of oligomannuronic acids and oligoguluronic acids by high-performance anion-exchange chromatography and electrospray ionization mass spectrometry

Oligomannuronic acids and oligoguluronic acids were prepared by enzymatic hydrolysis of alginate with alginate lyases. The oligosaccharides generated up to degree of polymerization 16 were characterized by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) and electrospray ionization mass spectrometry (ESI-MS). Acetate buffer linear gradients were used as mobile phases for separations of oligosaccharides. ESI-MS and HPAEC-PAD are very effective for the analysis and characterization of anionic oligosaccharides.     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers

The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.     

Structural analysis of oligosaccharides by a combination of electrospray mass spectrometry and bromine isotope tagging of reducing-end sugars with 2-amino-5-bromopyridine

Model reducing-end oligosaccharides were successfully labeled by a brominated aromatic amine reagent, 2-amino-5-bromopyridine (ABP), through reductive amination. Using either a combination of liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation or liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), sequence information corresponding to the model oligosaccharides was revealed with little ambiguity via the diagnostic unique twin peaks arising from the bromine isotopes, for both the molecular ions of the derivatized oligosaccharides and their fragments. No fragment ions arising from loss of the bromine atom were observed.     

系列甘露糖醛酸寡糖的制备与鉴定

用酸降解法制备了系列甘露糖醛酸寡糖(聚合度2～8),并分析测定了寡糖的结构. 褐藻胶经部分酸水解,于pH=2.85处分级获得聚甘露糖醛酸. 继续用酸降解法降解聚甘露糖醛酸,经凝胶柱层析分离纯化,获得系列甘露糖醛酸寡糖. 用荧光标记糖电泳(FACE)对寡糖进行了分析,并用电喷雾离子化质谱(ESI-MS)、 核磁共振波谱(NMR)及红外光谱(FTIR)进行了结构表征. 本研究用酸降解法制备饱和甘露糖醛酸寡糖,用凝胶柱层析法分离获得系列聚合度的寡糖,为褐藻胶大分子构效关系研究和药物的筛选与发现提供了重要的基础资料.     

Determination of linkages of linear and branched oligosaccharides using closed-ring chromophore labeling and negative ion trap mass spectrometry

Linear as well as branched oligosaccharides were labeled with p-aminobenzoic ethyl ester (ABEE) using the glycosylamine closed-ring labeling approach and analyzed by negative-ion electrospray ionization mass spectrometry (ESI-MS). Linkage specific fragment ions of ABEE labeled linear oligosaccharides were proposed based on the MS2 and MS3 data for several ABEE labeled linear oligosaccharides with known linkage configurations. Fragmentation at the reducing end was similar to that observed for ABEE disaccharides whereas the fragmentation pattern not involving the reducing end was similar to underivatized disaccharides. Based on these ions, all the linkages of linear oligosaccharides could be unambiguously determined. The fragmentation pattern at the branched sugar was in general not quite the same as the linear one. However, many linkage specific fragment ions were also observed for linkages at the branched sugar. These ions along with the ions proposed for linear oligosaccharides were found to be quite useful for the determination of all the linkages of branched oligosaccharides.     

A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants

Different separation (HPAEC, RP-HPLC, CE) and identification (MALDI-TOF-MS, ESI-MS(n)) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC-MALDI-TOF-MS) or online (CE-ESI-MS(n), RP-HPLC-ESI-MS(n)) connection provided complementary information on xyloglucan structure. Online CE-MS and RP-HPLC-MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains.     

Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.     

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.     

Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2-mers to 16-mers. Comparison with hyaluronic acid oligomers

This study reports for the first time the complete liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only in the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]2- product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There has been no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Unsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern as deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study, we were unable to distinguish between unsulfated chondroitin and HA.     

Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis

We report on microbore liquid chromatography (microLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to microLC/electrospray ionization/mass spectrometry (ESI-MS) and microLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man(7)GlcNAc(2) and Man(14)GlcNAc(2). Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-N-glycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man(9)GlcNAc(2), two for Man(10)GlcNAc(2), three for Man(11)GlcNAc(2), Man(12)GlcNAc(2), and Man(13)GlcNAc(2), and two for Man(14)GlcNAc(2). The CE results provided complementary information to the microLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.     

Electrospray ionization mass spectrometry fingerprinting of beer

After just simple degassing, dilution, pH adjustment and direct flow injection, characteristic fingerprint spectra of beer samples have been obtained by fast (few seconds) electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes. A total of 29 samples belonging to the two main beer types (lagers and ales) and several beer subtypes from USA, Europe and Brazil could be clearly divided into three groups both by simple visual inspection of their ESI(+)-MS and ESI(-)-MS fingerprints as well as by chemometric treatment of the MS data. Diagnostic ions with contrasting relative abundances in both the positive and negative ion modes allow classification of beers into three major types: P = pale (light) colored (pilsener, pale ale), D = dark colored (bock, stout, porter, mild ale) and M = malt beer. For M beers, samples of a dark and artificially sweetened caramel beer produced in Brazil and known as Malzbiers were used. ESI-MS/MS on these diagnostic beer cations and anions, most of which are characterized as arising from ionization of simple sugars, oligosaccharides, and iso-alpha-acids, yield characteristic tandem mass spectra adding a second and optional MS dimension for improved selectivity for beer characterization by fingerprinting. Direct ESI-MS or ESI-MS/MS analysis can therefore provide fast and reliable fingerprinting characterization of beers, distinguishing between types with different chemical compositions. Other unusual polar components, impurities or additives, as well as fermentation defects or degradation products, could eventually be detected, making the technique promising for beer quality control.     

Direct analysis of herbal powders by pipette-tip electrospray ionization mass spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) is widely used for analysis of solution samples. The development of solid-substrate ESI-MS allows direct ionization analysis of bulky solid samples. In this study, we developed pipette-tip ESI-MS, a technique that combines pipette tips with syringe and syringe pump, for direct analysis of herbal powders, another common form of samples. We demonstrated that various herbal powder samples, including herbal medicines and food samples, could be readily online extracted and analyzed using this technique. Various powder samples, such as Rhizoma coptidis, lotus plumule, great burdock achene, black pepper, Panax ginseng, roasted coffee beans, Fructus Schisandrae Chinensis and Fructus Schisandrae Sphenantherae, were analyzed using pipette-tip ESI-MS and quality mass spectra with stable and durable signals could be obtained. Both positive and negative ion modes were attempted and various compounds including amino acids, oligosaccharides, glycosides, alkaloids, organic acids, ginosensides, flavonoids and lignans could be detected. Principal component analysis (PCA) based on the acquired mass spectra allowed rapid differentiation of closely related herbal species.     

Separation and characterization of underivatized oligosaccharides using liquid chromatography and liquid chromatography-electrospray ionization mass spectrometry

Native cyclodextrin-based columns are particularly useful for the analysis of oligosaccharides because the retention of these carbohydrates is based mainly on the hydrogen bonding interactions of oligosaccharide hydroxyl groups with the stationary phase. Thus, the retention time predictably increases with the number of analyte hydroxyl groups, which corresponds to the elongation of the oligosaccharide chain. High-performance liquid chromatography (HPLC) coupled to electrospray ionization (ESI) mass spectrometry (MS) was used for the separation and characterization of underivatized oligosaccharide mixtures. With the limits of detection as low as 50 pg, all individual components of oligosaccharide mixtures (up to 11 glucose units long) were baseline resolved on a Cyclobond I 2000 column and detected using ESI-MS. Low flow rates and narrow I.D. columns increase the ESI-MS sensitivity significantly. The method showed potential usefulness for the sensitive and quick analysis of hydrolysis products of polysaccharides, and for trace levels of individual oligosaccharide or oligosaccharide isomers from biological systems.     

固相酸解法制备古糖酯寡糖及其电喷雾质谱分析

以褐藻酸中分离的聚古罗糖醛酸(PG)为原料, 以三氧化硫-吡啶为硫酸酯化试剂, 采用正交实验法确定了制备高硫酸酯基取代古糖酯(PGS)的最佳工艺条件, 并采用2D-NMR分析对其结构进行了确证. 本文建立了一种新的环境友好型固相酸解方法以制备PGS的寡糖(即采用732#阳离子交换树脂这种固态酸对PGS进行降解). 结果表明, 当732#阳离子交换树脂用量为200 mg/mL、PGS的质量分数为2%时, 在100 ℃下降解6 h可得到重均分子质量(Mw)小于3000的PGS寡糖, 经Bio-Gel P6凝胶层析柱分离可以得到13个聚合度单一的寡糖组分F1~F13. 电喷雾质谱(ESI-MS)分析结果表明, F1~F13分别是聚合度为1~13的PGS寡糖.     

Structural characterization of neutral oligosaccharide mixtures through a combination of capillary electrochromatography and ion trap tandem mass spectrometry

A CEC/ESI-MS/MS combined system has been developed for the separation and on-line structural analysis of neutral oligosaccharides. Various types of isomeric oligosaccharides were first successfully separated by CEC using polar monolithic columns, while the on-line tandem mass spectrometry has been explored to differentiate and elucidate the structures of isomeric oligosaccharides. The experimentally obtained tandem spectra usually provide sequence, branching, and linkage information. Oligosaccharide isomers with a different monomeric composition and branching showed different patterns of glycosidic linkage cleavage (B- and Y-ion series), allowing us to deduce their sequence and branching points. Isomers with different linkages were distinguished by identifying cross-ring fragment ions (A-ion series). While (1-->4) linkages yielded dominant (0,2)A ions, (1-->6) linkages showed an extensive and complete cross-ring cleavage series: (0,2)A, (0,3)A, and (0,4)A ions. Although the anomeric configurations and monosaccharide identification are rarely obtained from tandem MS, the relevant mixture components can be completely resolved with high-efficiency CEC columns featuring a polar functionality.     

Optimized conditions for high-performance liquid chromatography analysis of oligosaccharides using 7-amino-4-methylcoumarin as a reductive amination reagent

Reductive amination reaction using 7-amino-4-methylcoumarin (AMC) as a fluorescent probe enabled analyses of glycoproteins' monosaccharides and N-linked oligosaccharides. Reductive amination of N-acetylhexosamines and AMC using sodium cyanoborohydride or dimethylamine-borane complex indicated slight recovery of derivatives, but pyridine-borane achieved better recoveries. Reversed-phase high-performance liquid chromatography (HPLC) analyses of monosaccharides constituting glycoprotein glycans using fluorimetric detection revealed linearity for 0.2fmol to 1pmol, with less than 5% RSD quantitation reproducibility. Reversed-phase HPLC analyses of glycoprotein glycans, combined with negative-ion electrospray ionization mass spectrometry (LC-ESI-MS), enabled their structural determination. Using this highly hydrophobic reagent, AMC-labeled oligosaccharides displayed one-order to two-order higher ESI-MS intensity than derivatives labeled using other reagents.     

Nano-HPLC of Oligosaccharides – Method Development and Optimization

 The separation of homologous maltodextrins using methyl-, ethyl- and n-butyl esters of aminobenzoic acid and n-heptyloxyaniline by means of reversed phase nano-HPLC is presented. Fused silica capillaries packed with four different stationary phases were tested, the separation of the derivatized sugars was optimized and the separation properties of these columns were tested on the basis of the van Deemter plots. UV/VIS detection was used (285 nm for the aminobenzoic esters and 275 nm for the aniline derivatives) in combination with on-line ESI triple quadrupole mass spectrometry and off-line MALDI-TOF mass spectrometry. The choice of the stationary phase had a tremendous influence on the separation. Optimal results were obtained with an encapsulated ODS stationary phase and n-butyl aminebenzoate as label for the oligosaccharides. Malto-oligosaccharides with a degree of polymerization of up to of 25 could be resolved to baseline. On-line coupling with ESI-MS yielded additional information as a result of induced fragmentation and increased sensitivity by monitoring precursor ion. Off-line mass detection with MALDI-TOF was performed on the separated derivatized saccharides collected from nano-HPLC runs. The MALDI-TOF mass spectra confirm the ESI-MS data. Received January 25, 2001. Revision April 28, 2001.     

刺五加寡糖的电喷雾多级串联质谱研究

采用小柱层析法从刺五加中分离得到刺五加寡糖类系列化合物(刺五加二糖刺五加六糖).实验结果表明,在正离子模式下的ESI-MS谱中,此类化合物呈现出特征的加合离子峰簇[M+Na]⁺/[M+K]⁺或[M+H₂O+Na]⁺/[M+H₂O+K]⁺,可以确定其分子量;在负离子模式下的ESI-MS谱中,刺五加寡糖易形成[M-H]^-/[M+nH₂O-H]^-(n<3).还利用电喷雾多级串联质谱(ESI-MSⁿ)对刺五加三糖进行了系统的研究,推断出刺五加三糖的组成与结构.     

Quantitative determination of lysozyme-ligand binding in the solution and gas phases by electrospray ionisation mass spectrometry

Affinity constants for the binding of a range of substrate and non-substrate oligosaccharides to hen egg white lysozyme were determined by direct observation of the protein.ligand complexes using electrospray ionisation mass spectrometry (ESI-MS) with a chip-based nano-ESI source. The values obtained for a series of beta-1,4-N-acetylglucosamine oligomers (NAGn) were found to be in good agreement with those determined by fluorescence measurement. Oligomers of alpha-1,4-glucose (Glcn), which are believed to bind to lysozyme non-specifically, exhibited a 10(6)- to 10(8)-fold lower affinity for the enzyme. Lysozyme.NAGn complexes displayed an increase in Ka from n=2 to n=4, but then reached a plateau. In contrast non-specific lysozyme.Glcn complexes showed no such trend. Determination of gas-phase complex stability was achieved by quantitative collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) measurements. The collision energy (Ec50) or laser power (IRMPD50) required to dissociate precursor ions to 50% of their original intensity was determined for lysozyme.NAGn and Glcn complexes using the [M+8H]8+ charge state. An excellent correlation between trends in Ka and gas-phase stability was seen for NAGn oligomers bound to lysozyme, whereas no such relationship was observed with the non-specific, weaker lysozyme.Glcn complexes. These results illustrate that ESI-MS can be used to quantify the interactions between lysozyme and oligosaccharides in both the solution and gas phase and that measurement of gas-phase complex stability by CID or IRMPD can provide information about specific solution binding events.