基于电喷雾电离质谱(ESI-MS)的苯胺稳定同位素标记对还原性寡糖(链)的定性定量分析方法

建立了一种基于电喷雾电离质谱的苯胺稳定同位素标记对还原性寡糖链进行定性及相对定量分析的研究方法. 用苯胺标记乳糖标准品, 优化了影响标记效率的各种因素, 在弱酸性环境下, 选择糖链/苯胺/硼氢氰化钠的摩尔比为1∶1.2∶10, 于70 ℃反应15 min即可标记完全; 同时考察了4对d0/d5苯胺标记的麦芽糊精寡糖在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明, 在15倍动态范围内, 相对定量方法呈良好的线性关系(R=0.9986)和重现性(CV=10.20%). 为进一步验证定量方法的可靠性, 将其应用于人奶中游离寡糖(HMOs)和牛奶中游离寡糖(BMOs)的分析. 研究结果表明, 人奶中的乳糖含量高于牛奶, 人奶游离寡糖比牛奶游离寡糖种类复杂, 且岩藻糖基化程度高. 该方法成本低廉, 标记效率高且后处理方法简单方便, 适于微量样品通量化分析, 对差异糖组的研究有重要意义.     

The influence of sialylation on glycan negative ion dissociation and energetics

For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated.     

Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2‐aminoacridone

Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high‐voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2‐aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on‐capillary anionic borate‐polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ～50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.     

Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals

Carbohydrate chains in glycoprotein pharmaceuticals play important roles for the expression of their biological activities, but the structure and compositions of carbohydrate chains are dependent on the conditions for their production. Therefore, evaluation of the carbohydrate chains is quite important for productive process development, characterization of product for approval application, and routine quality control. The oligosaccharides themselves have complex structure including blanching and various glycosidic linkages, and oligosaccharides in one glycoprotein pharmaceutical generally have high heterogeneity, and characterization of oligosaccharide moiety in glycoprotein has been a challenging target. In these situations, CE has been realized as a powerful tool for oligosaccharide analysis due to its high resolution and automatic operating system. This review focuses on the application of CE to the glycoform analysis of glycoproteins and profiling of the N-linked glycans released from glycoprotein pharmaceuticals. Current applications for structure analysis using CE-MS(n) technique and glycan profiling method for therapeutic antibody are also described.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

Separation of Reducing Oligosaccharides Derived from Glycoproteins on Stable Polymeric HPLC Packings

Abstract

A multidimensional high performance liquid chromatographic approach has been designed to purify and isolate oligosaccharides derived from glycoproteins prior to structure elucidation. After release by chemical or enzymatic means, crude oligosaccharide mixtures can be fractionated into acidic and neutral glycans by using a stable polymeric anion exchange column, Glyco-Pak DEAE. Acidic fractions can be further separated on the same column by changing mobile phase selectivity. Neutral oligosaccharides are resolved using partition mode chromatography first on a silica-based Carbohydrate Analysis column followed by a hydroxylated polymeric Glyco-Pak N column. Crude mixtures containing only neutral glycans can be resolved directly on Glyco-Pak N. These stable polymeric columns do not shed packing materials which may interfere with post-column structural analyses such as NMR. This multicolumn strategy is demonstrated by using acidic and neutral oligosaccharides derived from hydrazinolysis of fetuin and ovomucoid respectively and by using high mannose compounds released by Endohexosaminidase H treatment as examples which support this approach.     

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 μL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex₂Fuc₂ and Hex₃HexNAc₁Fuc₂, as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a − b+), Le(a + b−), and Le(a − b−), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.     

Application of 2,3-naphthalenediamine in labeling natural carbohydrates for capillary electrophoresis

Neutral and acidic monosaccharide components in Ganoderma lucidum polysaccharide are readily labeled with 2,3-naphthalenediamine, and the resulting saccharide-naphthimidazole (NAIM) derivatives are quantified by capillary electrophoresis (CE) in borate buffer. Using sulfated-α-cyclodextrin as the chiral selector, enantiomers of monosaccharide-NAIMs are resolved on CE in phosphate buffer, allowing a simultaneous determination of the absolute configuration and sugar composition in the mucilage polysaccharide of a medicinal herb Dendrobium huoshanense. Together with the specific enzymatic reactions of various glycoside hydrolases on the NAIM derivatives of glycans, the structures of natural glycans can be deduced from the digestion products identified by CE analysis. Though heparin dissachrides could be successfully derived with the NAIM-labeling method, the heparin derivatives with the same degree of sulfation could not be separated by CE.     

寡糖—8—氨基芘—1,3,6—三磺酸衍生物的毛细管电泳—激光诱？…

利用自组装的毛细管电泳－激光诱导荧光装置,研究了多种寡糖－８－氨基芘－１,３,６－三磺酸（寡糖ＡＰＴＳ）衍生物的分离。考察了电泳介质、浓度及pＨ对寡糖－ＡＰＴＳ 衍生物分离的影响,在酸性和碱性条件下,分别实现了痕量寡糖标准品及葡聚糖水解产物的高效分离。     

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

Resolution of structural isomers of sialylated oligosaccharides by capillary electrophoresis

The resolution of structural isomers in mixtures of oligosaccharides is often challenging. Capillary electrophoresis was employed to separate three sets of structural isomers of sialylated oligosaccharides found in human milk and bovine colostrum. Different running buffers were necessary to achieve optimal baseline resolution. To resolve 3'- and 6'-sialyllactoses, 0.2 M aqueous sodium phosphate containing 40% methanol as an organic modifier was used as a running buffer. To resolve 3'- and 6'-sialyllactosamines, 0.4 M aqueous sodium phosphate without organic modifier was used. Baseline resolution of sialyllacto-N-tetraose-a and -b and sialyllacto-N-neotetraose-c was achieved with a 0.4 M Tris-HCl buffer containing 250 mM sodium dodecyl sulfate and 10% methanol as the organic modifier. Thus, each of these sets of structural isomers of sialylated oligosaccharides required a unique running buffer with respect to buffer type, concentration, pH, presence of organic modifiers, and surfactants. Similar electrophoresis conditions may be useful for resolving and analyzing other structural isomers of acidic oligosaccharides by capillary electrophoresis.     

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.     

Comparative studies on the analysis of urinary trypsin inhibitor (ulinastatin) preparations

Urinary trypsin inhibitor (ulinastatin) is a characteristic protein pharmaceutical which contains both glycosaminoglycans and N-linked glycans in its molecule and has been used for treatment of acute pancreatitis. The comparability of ulinastatin preparations of different lots or from different companies was studied by using conventional analytical approaches such as SDS-PAGE, cellulose acetate membrane electrophoresis, and HP size-exclusion chromatography (SEC) and also by using newly developed techniques such as CE and MALDI-TOF MS. The methods using SEC and SDS-PAGE according to The Japanese Pharmacopoeia showed similar molecular masses for two different preparations, and the estimated molecular masses were significantly different from those observed with MALDI-TOF MS. We also showed that the electrophoretic methods using cellulose acetate membrane electrophoresis and CE can be used for comparability assessments of ulinastatin preparations. In addition, we analyzed the unsaturated disaccharides derived from glycosaminoglycan (chondroitin 4-sulfate chain) and N-linked oligosaccharides attached to ulinastatin by CE after releasing them by enzymatic digestion followed by fluorescent labeling with 2-aminoacridone and 2-aminobenzoic acid, respectively. The results indicated that carbohydrate chains are important as markers for comparability assessments of ulinastatin pharmaceutical preparations.     

Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.     

Mass spectrometric study of N‐glycans from serum of woodchucks with liver cancer

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N‐glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures – reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and solid‐phase extraction (SPE) on a cation‐exchange/reversed‐phase STRATA‐XC cartridge – were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N‐glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N‐glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N‐glycans from total serum in the progression of liver disease. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation of the major O-glycosidically linked oligosaccharides obtained by alkaline borohydride degradation of human meconium glycoproteins

Neutral and acidic oligosaccharides derived from human meconium glycoproteins by alkaline borohydride degradation have been separated by high-performance liquid chromatography on a Micro-Pak anion-exchange column. In each class, oligosaccharides were purified by normal-phase (neutral and acidic oligosaccharides) and reversed-phase (neutral oligosaccharides) chromatography. Effective separations of neutral oligosaccharides and acidic oligosaccharides were achieved.     

Nanoliquid chromatography-mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high-accuracy mass analyzer

The nanoLC separations of oligosaccharides using microchip-based columns are described. Mixtures of alditols from mucins and human milk are separated on graphitized carbon. The nanoLC-MS device showed high mass accuracy for the oligosaccharides ranging between 1 and 6 ppm on routine analyses. The high mass accuracy readily allowed identification of oligosaccharide peaks and the determination of their compositions. High retention time reproducibility was exhibited by the microchip LC. Little variation was observed for standard sample either alone or in a complex heterogeneous mixture. The nanoLC-MS exhibits excellent capabilities in profiling mixtures of oligosaccharides.     

Highly sensitive capillary electrophoresis analysis of N-linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser-induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).     

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.     

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.