On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins

An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization.     

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision‐induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra‐cyclic C‐6 carbon of hexoses or N‐acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged ‐SO₃^– residue attached to the oligosaccharide precursor becomes protonated by a CID‐induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M – 2H]^2– ions or the sodiated [M + Na – 2H]^– ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M – H]^– ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High‐Energy C‐trap Dissociation) in an Orbitrap mass spectrometer. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

山茱萸炮制过程中环烯醚萜苷类成分的质谱研究

利用高效液相色谱-电喷雾多级串联质谱(HPLC-ESI-MSn)联用技术, 对传统中药山茱萸炮制过程中环烯醚萜苷类成分的变化进行了研究. 采用反相C18色谱柱, 二元线性梯度洗脱, 分离并获得了山茱萸中7个环烯醚萜苷类化合物. 并通过电喷雾一级质谱获得了上述7种化合物的分子量信息, 利用电喷雾质谱的源内碰撞诱导解离技术, 获得了该类化合物在负离子模式下的碎裂特征, 在此基础上, 对其进行了结构鉴定和含量分析. 首次发现了差向异构体7α-乙氧基莫诺苷和7β-乙氧基莫诺苷化合物. 研究结果表明, 当采用HPLC-ESI-MS法分析山茱萸环烯醚萜苷类化合物时, 通过色谱保留时间色谱峰面积和质谱特征两方面信息能够提供更加准确可靠的定性定量结果.     

Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry

The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: [structure: see text] After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids.     

Diagnostic ions for the rapid analysis by nano-electrospray ionization quadrupole time-of-flight mass spectrometry of O-glycans from human mucins

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was used for sensitive mapping and sequencing of underivatized oligosaccharide alditols obtained from human mucins. Using subnanomolar amounts of oligosaccharides previously analyzed by nuclear magnetic resonance (NMR), series of diagnostic ions relevant to the structural characterization of O-glycans were deduced. Determination of the core type as well as positions and partial linkages of fucose residues could be readily obtained from the dominant [M+Na](+) ions. Differentiation of isomeric structures and glycosidic linkages were defined by the characteristic cross-ring (0,2)A-type cleavages in the negative ion mode. Tandem (MS/MS) mass spectra of [M-H](-) ions from sialylated or sulfated O-glycans revealed information concerning the position and linkage of such residues. These fragmentation rules were further applied in the structural determination of glycans from human colonic mucins. All these findings indicated the efficiency of ESI-Q-TOFMS for the determination of oligosaccharide composition, sequence, partial linkage and substitution, providing a wealth of structural information with sensitivity sufficient for the analysis of quantities as obtained from natural sources.     

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.     

Matrix-assisted laser desorption/ionization in-source decay combined with tandem time-of-flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in-source decay (ISD) with the subsequent TOF/TOF-MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second-generation fragment ions were different from those observed in conventional MALDI-TOF/TOF-MS experiments. Other fragments, which had already been observed in conventional MALDI-TOF/TOF-MS and again showed up in second-generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross-ring fragmentation by high-energy collision-induced fragmentation. In conclusion, combination of MALDI-ISD with TOF/TOF-MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics.     

Gas chromatography and gas chromatography/mass spectrometry for the characterization of complex mixtures of large oligosaccharides

High temperature gas chromatography and gas chromatography/mass spectrometry are used in the characterization of complex natural mixtures of permethylated oligosaccharides released from proteins or lipids. The high resolution allows separation of isomeric compounds and the mass range extends to oligosac-charides around molecular mass 2000 daltons or 10 sugar residues for isomalto-oligosaccharides. The mass spectra of permethylated oligosaccharide alditols from mucin glycopeptides are very informative and the approach allows a simple and rapid characterization of these complex components.     

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.     

高效液相色谱-电喷雾质谱联用法测定人参和西洋参的皂苷类成分

利用高效液相色谱（LC—ELSD）与电喷雾质谱（ESI—MS）联用技术对人参和西洋参中的人参皂苷类成分进行了比较研究。通过液相色谱与质谱联用技术获得了相应化合物的分子量信息;利用质谱的源内CID技术获得了相应化合物的结构信息。根据人参皂苷Rf和拟人参皂苷F11两种同分异构体在质谱中的源内CID裂解规律的不同,建立了人参和西洋参药材的LC-ESI-MS简便、准确的鉴别方法。     

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.     

Linkage and branch determination of N-linked oligosaccharides using sequential degradation/closed-ring chromophore labeling/negative ion trap mass spectrometry

A method based on sequential degradation, p-aminobenzoic ethyl ester (ABEE) closed-ring labeling, and negative ion electrospray ionization tandem mass spectrometry is presented for the study of linkage and branch determination for N-linked oligosaccharides. Closed-ring labeling provides greater linkage information than the more popular open-ring reductive amination approach. In addition, after high-performance liquid chromatography (HPLC) separation, closed-ring labeling allows for regeneration of the underivatized oligosaccharide, a requirement for alkaline sequential degradation. The analytical scheme presented here uses HPLC separation of closed-ring labeled oligosaccharides to resolve the mixture into individual forms that undergo subsequent structural analysis by negative ion tandem mass spectrometry. To facilitate complete structural analysis, particularly for larger sugars, the closed-ring labels are removed and the sugars are sequentially degraded by controlled alkaline hydrolysis. It is noteworthy that for sugars containing sialic acid moieties, a protecting group must be used to stabilize sialic acid groups during sequential alkaline degradation. This described approach was applied to two high mannose oligosaccharides M5G2, M6G2 cleaved from the ribonuclease B and a complex oligosaccharide A2 cleaved from transferrin.     

Analysis of oversulfation in a chondroitin sulfate oligosaccharide fraction from bovine aorta by nanoelectrospray ionization quadrupole time-of-flight and fourier-transform ion cyclotron resonance mass spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions.     

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans

We have prepared a number of isomeric red seaweed galactan-derivative sulfated oligosaccharides to determine whether there were diagnostic differences among the isomeric mass spectra obtained using ESI CID MS/MS (triple quadrupole instrument). Fragmentation of the single or multicharged molecular ions from di-, tetra-, and hexasaccharides indicated that the relative positioning of the sulfate groups and type of monosaccharide unit affect the rate of cleavage of the glycosidic bonds. We also performed a comparative [M-Na]⁻ fragmentation study of positional isomers of sulfated disaccharides that present all four monosulfation possibilities on the galactopyranosidic ring. In this case, negative-ion ESI CID MS/MS approach gave diagnostic product ions from cross-ring cleavages along with the same main B₁ ion (from sulfated Gal_p), at m/z 241, for all isomers. The isomeric disaccharides were also submitted to increased spray energy conditions inducing in-source fragmentation; preformed B₁ ions were then fragmented to give similar product ions as those found in [M-Na]⁻ analysis. Evaluation of the relative abundances mainly for cross-ring fragment ions at m/z 138, 139, 151, 153 allowed clear distinction among the members of the disaccharide series. The different ratios for m/z 151/153 ions were consistent with the predominance of m/z 153 being related to the cases when the bond involved in the cleavage process links a sulfated carbon. A quadrupole ion trap instrument (MSⁿ analysis) was also utilized to compare the results obtained with the triple quadrupole instrument.     

高效液相色谱-质谱联用分析无患子中的表面活性物质

 应用高效液相色谱和大气压电离质谱联用技术 ,分离分析了无患子果皮中的表面活性成分。根据质谱结果确定其相对分子质量 ,根据源内的碰撞诱导解离 (CID)技术产生的碎片初步推测表面活性物质的结构 ,发现了数个未见文献报道的组分。     

MALDI-TOF and ESI-MS analysis of oligosaccharides labeled with a new multifunctional oligosaccharide tag

A new multifunctional oligosaccharide label with a 1 degree amino-group was synthesized and characterized. The oligosaccharide label was introduced into several neutral oligosaccharides by reductive amination, and the derivatives were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and by electrospray ionization (ESI) mass spectrometry. It was demonstrated that the labeling reaction was satisfactory, and that as little as 50 pmol of starting material could be efficiently labeled with minimal loss to side reactions. A mixture of high-mannose N-glycans released from ribonuclease B was labeled. The label did not appear to interfere with structural characterization of the oligosaccharides by mass spectrometry. N-quaternization of the labeled oligosaccharides resulted in significantly increased sensitivity of detection with as little as 100 fmol on the probe detected. Deuterium coding of labeled oligosaccharide mixtures and relative abundance of mixture components was investigated. A protocol for the chromatographic separation of mixtures of labeled oligosaccharides by HPLC was developed and is reported here.     

On resin synthesis of sulfated oligosaccharides

Sulfated glycans are involved in many biological processes, making well-defined sulfated oligosaccharides highly sought molecular probes. These compounds are a considerable synthetic challenge, with each oligosaccharide target requiring specific synthetic protocols and extensive purifications steps. Here, we describe a general on resin approach that simplifies the synthesis of sulfated glycans. The oligosaccharide backbone, obtained by Automated Glycan Assembly (AGA), is subjected to regioselective sulfation and hydrolysis of protecting groups. The protocol is compatible with several monosaccharides and allows for multi-sulfation of linear and branched glycans. Seven diverse, biologically relevant sulfated glycans were prepared in good to excellent overall yield.

Well-defined sulfated oligosaccharides are important synthetic targets. We present an on resin approach for the synthesis of sulfated glycans with a broad reaction scope that overcomes previous limitations associated with on resin synthesis.     

Characterization of a gel-separated unknown glycoprotein by liquid chromatography/multistage tandem mass spectrometry: analysis of rat brain Thy-1 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.     

Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.