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。     

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.     

Microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization

A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 μm) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins.     

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.     

Capillary electrophoretic separation of heparin oligosaccharides under conditions amenable to mass spectrometric detection

A capillary electrophoresis method for the separation of high-molecular-mass heparin oligosaccharides compatible with mass spectral detection was developed. Structurally defined heparin oligosaccharides ranging in size from tetrasaccharide to tetradecasaccharide were used to optimize the conditions. Applying normal and reversed polarity modes, these oligosaccharides were separated by CE under various conditions. Ammonium hydrogencarbonate (30 mM at pH 8.50) used as the running electrolyte system gave good separation efficiency and resolution in the normal polarity mode. Application of this method to the separation of complicated heparin oligosaccharide mixtures required the addition of electrolyte additives. Ammonium hydrogencarbonate (30 mM), containing triethylamine (10 mM), was useful for the separation of complex oligosaccharide mixtures. Run-to-run and day-to-day precision and limits of detection were established for these separations.     

Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9?×?250 mm) ion exchange column with sodium chloride gradient elution and UV detection at 232 nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions.

Hydrophilic interaction liquid chromatography for the separation,purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz

A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid‐phase extraction followed by hydrophilic interaction liquid chromatography at semi‐preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible.     

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.     

Capillary electrophoresis coupled to electrospray mass spectrometry through a coaxial sheath flow interface and semi-permanent phospholipid coating for the determination of oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonic acid

For the first time, a semi-permanent phospholipid coating is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). Although phospholipids in free solution are generally avoided in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with α-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme β1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin.     

Fractionation of glycoprotein-derived oligosaccharides by affinity chromatography using immobilized lectin columns

Lectin affinity column chromatography is becoming a method of choice for the fractionation and purification of oligosaccharides, especially N-linked oligosaccharides. Using lectin affinity, it is easy to separate structural isomers and to isolate oligosaccharides based on specific features. Further, serial lectin column chromatography, when various lectin columns are used at the same time, can afford a very sensitive method for the fractionation and characterization of extremely small amounts of oligosaccharides. Thus, when used in conjunction with other separation techniques, lectin affinity chromatography can help to purify rapidly oligosaccharides and provide substantial information about their structural features.     

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.     

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.     

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.     

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.     

Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure

A capillary electrophoresis (CE) method was developed for the simultaneous analysis of small chitin and chitosan oligosaccharides. For detection purposes, the oligomers were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), a well known fluorophore for oligosaccharides analysis. The detection was performed by laser-induced fluorescence (LIF) with an argon ion laser having an excitation wavelength of 488 nm and with emission monitored at 520 nm. Derivatization parameters such as reaction time and conditions were examined. Separation conditions were also varied by testing a range of buffer pHs and concentrations. The best conditions were found using an 80 mM borate buffer at pH 8.4. This CE-LIF optimized method was used for the analysis of an enzymatically produced oligo-chitosan sample composed of a complex mixture and having an average degree of polymerization of 3.7 monomer units and 80% deacetylation. The oligo-chitosan sample was treated with a chitin deacetylase-like enzyme, the products were derivatized with APTS, and then analyzed without purification. The goal was to determine whether the deacetylase-like enzyme could increase the extent of deacetylation of the oligo-chitosan sample.     

Ultrafast analysis of oligosaccharides on microchip with light-emitting diode confocal fluorescence detection

We have developed a new method for the high-speed separation and high-sensitivity detection of complex oligosaccharides based on microchip electrophoresis (nu-CE) with light-emitting diode (LED) confocal fluorescence detection. Oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) were found to strongly adsorb to the surface of polymethylmethacrylate (PMMA) microchips. Accordingly, three classes of major dynamic coating additives were systematically investigated, and cellulose derivatives were found to specifically suppress such adsorption and allow high-performance separation on PMMA chips. Additive concentration, buffer pH and applied field strength were found to be key factors in the high-performance separation& of APTS-labeled oligosaccharides on PMMA chips. Under optimal conditions, 15 oligosaccharides in dextrin hydrolysate can be separated within 45 s with an electrophoretic separation efficiency of over 400 000 theoretical plates per meter. The relative standard deviation (RSD) values of migration times of fourteen oligosaccharides were less than 0.50% between six different channels, and the detection limit for APTS-labeled glucose was about 1.98 x 10(-8) mol/L or 8.61 amol with a signal-to-noise ratio (S/N) of 3. The high speed, high efficiency and high sensitivity of this micro-CE-based method indicate that it can be widely applied to analysis of complex oligosaccharides.     

κ-卡拉胶寡糖的反相离子对-超高效液相色谱-四极杆-飞行时间质谱研究

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

Structural Characterization of Neutral Oligosaccharides by Laser-Enhanced In-Source Decay of MALDI-FTICR MS

MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1 → 4)-, α(1 → 6)-, β(1 → 4)-, and β(1 → 3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.     

Electrochemical Glycosylation as an Enabling Tool for the Stereoselective Synthesis of Cyclic Oligosaccharides

Electrochemical glycosylation of a linear oligosaccharide with a protecting-group-free primary hydroxyl group afforded cyclic oligo-saccharides, up to hexasaccharides, in high yields. Precursors of the cyclic oligosaccharides were prepared by automated electro-chemical assembly-a method for the automated electrochemical solution-phase synthesis of oligosaccharides. We demonstrated that electrochemical glycosylation is useful not only for intermolecular glycosylation but also for intramolecular glycosylation to synthesize cyclic oligosaccharides.     

Reverse thin layer method for enhanced ion yield of oligosaccharides in matrix‐assisted laser desorption/ionization

A sample preparation method that is suitable for sensitive detection of underivatized oligosaccharides by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) has been investigated. As compared with the conventional dried‐droplet or ethanol (EtOH) recrystallization method, superior mass spectra in terms of ion yield and signal‐to‐noise (s/n) ratio were obtained when methanol (MeOH) was used as a solvent for the mixture of matrix and oligosaccharides. Based on these results, a new sample preparation method, named the ‘reverse thin layer method’, was developed. This method comprises two steps: first, complete drying of the oligosaccharide solution on the MALDI target plate; and second, deposition of the matrix dissolved in a small amount of MeOH. Using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained from oligosaccharides compared with conventional methods. Notably, the method can be applied to various matrices including both solid and liquid matrices. Copyright © 2009 John Wiley & Sons, Ltd.