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.     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。     

Rhodamine B amine as a highly sensitive fluorescence derivatization reagent for saccharides in reversed-phase liquid chromatography

6-Rhodamine B amine functions as a highly sensitive fluorescence derivatization reagent for mono- and oligosaccharides; it reacts with the reducing end of saccharides under acidic conditions. The fluorescent derivatives of five monosaccharides can be separated within 25 min by reversed-phase liquid chromatography with isocratic elution. The detection limits (S/N = 3) for mono-, di-, and oligosaccharides are 7-51, 13, and 9-35 fmol/20 microl injection, which correspond to analyte concentrations of 35-255, 65, 45-175 nM, respectively. We have applied this derivatization method successfully to the analysis of the components of oligosaccharides in glycoproteins (ribonuclease B and fetuin) following their acidic or enzymatic hydrolysis. The results from these analyses are in good agreements with the reported values established previously.     

Ultraviolet photodissociation of chromophore-labeled oligosaccharides via reductive amination and hydrazide conjugation

The fragmentation patterns of hydrazide-conjugated and reductively aminated oligosaccharides, including lacto-N-fucopentaoses and lacto-N-difucohexaoses, produced on collisionally induced dissociation (CID) and ultraviolet photodissociation (UVPD) in a quadrupole ion trap are presented. The two derivatization methods generate different cross-ring cleavages on UVPD and CID. UVPD of hydrazide-conjugated oligosaccharides yield predominant (2, 4)A-type cross-ring cleavage ions. In contrast, UVPD of aminated oligosaccharides results mainly in (0, 1)A-type ions. Moreover, more extensive dual-cleavage pathways (i.e. internal fragment ions) were observed on UVPD.     

Mass spectrometric analysis of carbohydrates labeled with a biotinylated tag

A derivatization method for mass spectrometric analysis of oligosaccharides is presented. Small saccharides, complex, high‐mannose‐type oligosaccharides and oligosaccharides released from hen ovalbumin were converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (BACH). Improved sensitivity of mass spectrometric analysis of labeled glycans in comparison with their natural counterparts was achieved after derivatization. The labeling reagent contains a biotin handle at one end and a hydrazide group at the other. Hence, the key feature of biotinylated sugars is that in addition to their usefulness in functional studies (e.g. analysis of the interaction between lectins and biotin‐derivatized oligosaccharides) they might be utilized also for structural analysis of oligosaccharides. Mass spectrometric studies were performed by matrix‐assisted laser desorption/ionization time‐of‐flight and electrospray ionization mass spectrometry. Copyright © 2009 John Wiley & Sons, Ltd.     

Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.     

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.     

Malononitrile as a new derivatizing reagent for high-sensitivity analysis of oligosaccharides by electrospray ionization mass spectrometry

A new method for the high-sensitivity analysis of oligosaccharides by negative ion electrospray ionization mass spectrometry was developed through a chemical derivatization of oligosaccharides. Oligosaccharides were derivatized to dinitrile compounds from the reaction with malononitrile under mildly basic conditions. The derivative of maltoheptaose was detected mainly as the [M-2H]2- ion in negative ion mode with 20 fmol sensitivity, even in unpurified samples. In this malononitrile derivatization method, no inorganic reagent, other than sodium hydroxide as a base catalyst, is used. Also, because excess ligand (malononitrile) is volatile, high sensitivity detection is realized without any solvent extraction or chromatographic purification. The detection limit can also be decreased by simple on-line cartridge filtration to 200 attomol which is 10(5) times better than that of free maltoheptaose. Structural information for oligosaccharide derivatives was obtained by collision induced dissociation. This malononitrile derivatization method is convenient and efficient for the sensitive analysis of oligosaccharides.     

LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.     

Quantitative analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry, capillary electrophoresis and anion exchange chromatography

The analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. Appropriate methods were developed which enabled the resolution of the oligosaccharides and quantification of the peak areas. It could be shown that each technique provided a different distribution profile of the maltodextrins. Using MALDI-TOF MS signals of higher molecular weight oligomers were enhanced while low molecular weight analogues were discriminated. Thus, the response factor depends on the degree of polymerization (DP) of the carbohydrates. Homologues up to DP-15 could be detected. Analysis of the maltodextrins by CE was accomplished by derivatization of the sugars with 4-aminobenzonitrile (ABN) and 8-aminonaphthalene-1,3,6-trisulfonic acid, respectively. By using the latter reagent oligosaccharides up to DP-13 were detected while derivatization with ABN allowed detection up to DP-9. The molecular weight distribution obtained by both approaches were the same. HPAEC-PAD enabled the determination of oligomers up to DP-9. The distribution obtained by this technique showed somewhat lower signals of the small homologues than those found by CE while the opposite held for higher molecular weight compounds. Hydrolysis of the carbohydrates by the derivatization reaction prior to CE analysis, which increased the proportion of low molecular weight homologues, may account for these findings.     

Direct and stereoselective synthesis of 1,3-cis-3- arylsulphonaminodeoxydisaccharides and oligosaccharides

The 3-aminoglycosides are ubiquitous in biologically important classes of glycoconjugates and naturally occurring oligosaccharides. Despite the rapid growth in the development of synthetic method of 3-amino glycosides, the current state-of-the art suffers from limited substrate scope, low yields, long reaction times, and anomeric mixtures. This work presents a novel direct method for the synthesis of 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides via α-selective glycosylation and hydroamination of glycal in a one-pot manner. This efficient multicomponent reaction methodology provides ready access to 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides and allows derivatization by variation of each component.     

Quantitative analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry, capillary electrophoresis and anion exchange chromatography

The analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. Appropriate methods were developed which enabled the resolution of the oligosaccharides and quantification of the peak areas. It could be shown that each technique provided a different distribution profile of the maltodextrins. Using MALDI-TOF MS signals of higher molecular weight oligomers were enhanced while low molecular weight analogues were discriminated. Thus, the response factor depends on the degree of polymerization (DP) of the carbohydrates. Homologues up to DP-15 could be detected. Analysis of the maltodextrins by CE was accomplished by derivatization of the sugars with 4-aminobenzonitrile (ABN) and 8-aminonaphthalene-1,3,6-trisulfonic acid, respectively. By using the latter reagent oligosaccharides up to DP-13 were detected while derivatization with ABN allowed detection up to DP-9. The molecular weight distribution obtained by both approaches were the same. HPAEC-PAD enabled the determination of oligomers up to DP-9. The distribution obtained by this technique showed somewhat lower signals of the small homologues than those found by CE while the opposite held for higher molecular weight compounds. Hydrolysis of the carbohydrates by the derivatization reaction prior to CE analysis, which increased the proportion of low molecular weight homologues, may account for these findings. Receiverd: 28 August 1997 / Revised: 24 November 1997 / Accepted: 25 November 1997     

Sequencing and characterization of oligosaccharides using infrared multiphoton dissociation and boronic acid derivatization in a quadrupole ion trap

A simplified method for determining the sequence and branching of oligosaccharides using infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. An IR-active boronic acid (IRABA) reagent is used to derivatize the oligosaccharides before IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. We demonstrate this approach for two series of oligosaccharides, the lacto-N-fucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).     

Reductive amination of carbohydrates using NaBH(OAc)<Subscript>3</Subscript>

An improved protocol for reductive amination of carbohydrates is developed. This derivatization facilitates the detection of oligosaccharides in HPLC-UV and mass spectrometric applications by enhancing the signal of the carbohydrates. In this study, reductive amination was achieved using NaBH(OAc)₃.This reducing agent is an attractive alternative to the toxic, but extensively used reducing agent, NaBH₃CN. Several types of carbohydrates were successfully derivatized using NaBH(OAc)₃, and the results obtained from this protocol were compared with those obtained with NaBH₃CN. Both reducing agents were equally effective in side-by-side analysis. Two purification strategies (purification by zip-tip and HPLC) were implemented and the instrumental limit of detection of each method was compared. The detection limit was ~1,000 times lower when the purification was done using HPLC, compared to using the zip-tip. Since the derivatization by-products in this protocol are not toxic, MS analysis also could also be performed directly, without purification. The MS/MS data of derivatized and underivatized oligosaccharides were acquired as well. The derivatized oligosaccharides produce more easily interpretable product ions than underivatized oligosaccharides.     

寡糖衍生化及基质辅助激光解吸电离飞行时间质谱分析方法研究

为提高中性寡糖在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)中的检测灵敏度,建立了以1-(4-氰基苯基)-4-哌啶碳酰肼(CPH)为衍生化试剂对寡糖的标记方法。寡糖的还原端与CPH的酰肼基团反应生成腙,使得寡糖被CPH标记,衍生物以MALDI-TOF质谱进行分析。结果表明:在反应温度95℃,醋酸浓度为0.125%(V/V),CPH过量100倍的条件下,衍生产率可达最大,并且CPH衍生可使中性寡糖在MALDI-TOF质谱中的检测灵敏度提高10倍。本方法简便快速,灵敏度高,适合微量寡糖链的质谱分析。     

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.     

Nano-HPLC-mass spectrometry and MEKC for the analysis of oligosaccharides from human milk

The separation of oligosaccharides derivatized with various esters of aminobenzoic acid by means of reversed-phase nano-HPLC (nHPLC) with on-line ESI mass spectrometry and off-line MALDI-TOF mass spectrometry as well as MEKC is described. For this purpose methyl, ethyl and butyl aminobenzoates and heptyloxyaniline were used as derivatization agents for homologous maltodextrins and oligosaccharides from human milk. Four different C(18) stationary phases were tested for this purpose because the type of stationary phase was shown to have a dramatic effect on the performance of the separation. Optimal results were obtained using n-butyl aminobenzoate as label and an encapsulated ODS stationary phase. The on-line coupling of nHPLC to ESI MS allowed to separate and identify various oligosaccharides from human milk. This technique enabled the exact attribution of the molecular structure to a signal in the chromatogram. In a second approach oligosaccharides were separated by nHPLC and subsequently fractionated. The fractions were analyzed by MALDI-TOF mass spectrometry. The results obtained by this approach confirmed the ESI MS data. An analogous separation profile was obtained by using sodium dodecyl sulfate in MEKC, which proves that the retention mechanisms of both techniques are identical.     

Preparative purification of tyrosinamide N-linked oligosaccharides

N-linked oligosaccharides from glycoproteins can be either analyzed on a sub-nanomole scale or preparatively purified on a multi-micromole scale. Each goal necessitates a unique analytical strategy often involving oligosaccharide derivatization to enhance separation and detection. Tyrosinamide-oligosaccharides were developed to facilitate the preparative purification of N-linked oligosaccharides. These have found many uses in oligosaccharide remodeling, in the preparation of neoglycoconjugates, in developing receptor probes, and even as analytical standards in chromatography. This review discusses progress in the preparation of tyrosinamide-oligosaccharides from different glycoproteins and their utility in glycobiology research.     

1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化寡糖链的HPLC分离及其激光解吸电离质谱分析

以1-苯基-3-甲基-5-吡唑啉酮(PMP)为衍生化试剂对寡糖链进行标记, 用氨水替代氢氧化钠溶液作碱性介质, 衍生化反应后氨水可通过干燥除去, 省去了脱盐处理过程, 衍生化的寡糖可直接进行激光解吸电离质谱分析. 建立起了PMP衍生化寡糖的RP-HPLC分离分析模式, 在此HPLC分析条件下, 可以对标记的寡糖链进行样品分离及制备.     

Analysis of carbohydrates by HPLC with post-column derivatization

Post-column derivatization of oligosaccharides after HPLC separation with a variety of systems has been achieved either by reaction with thymol in concentrated sulfuric acid or after hydrolysis of saccharides with hydrochloric acid and derivatization of the resulting monosaccharides with p-aminobenzoic acid hydrazide. With both reactions the detection of reducing and non-reducing sugars is possible even at trace levels. The latter reaction, despite the two reaction steps required, is much more convenient and practical, whereas with the first reaction it is also possible to determine alkylpolyglucosides.