气相色谱-质谱联用同时分析新型细菌多糖中的单糖和糖醛酸

对纯化的新型细菌多糖进行酸水解,用乙硫醇-三氟乙酸和醋酐-吡啶体系先后对酸水解物进行衍生,与之前报道不同的是糖醛酸得到有效衍生化。以木糖为内标,采用气相色谱-质谱联用(GC-MS)定量分析该多糖酸水解物中单糖和糖醛酸衍生物发现,该多糖的糖链由岩藻糖、葡萄糖、葡萄糖醛酸和半乳糖组成,其相对物质的量比为1.50:1.0:0.79:2.06;中性糖比例与糖醇乙酸酯化分析岩藻糖、葡萄糖和半乳糖的相对物质的量比(1.76:1.0:1.98)接近;糖醛酸咔唑法与该方法分析葡萄糖醛酸的含量分别为16.19%和14.85%。以上结果表明所建立的衍生化方法及GC-MS同时定量分析多糖酸水解物中单糖和糖醛酸的方法可行。此外还对葡萄糖醛酸的质谱裂解机理进行了阐述。     

柱前衍生高效液相色谱法分析海洋褐藻多糖药物的糖醛酸组成

采用三氟乙酸(TFA)分别将藻酸双酯钠(PSS)、甘糖酯(PMS)和古糖酯(PGS)3种海洋褐藻多糖药物进行降解,再与1-苯基-3-甲基-5-吡唑啉酮(PMP)进行柱前衍生,建立了3种药物糖醛酸组成分析的高效液相色谱方法.结果表明:PSS、PMS、PGS的最适降解条件:在110 ℃下降解6 h,TFA浓度3 mol/L;最适衍生条件:反应温度70 ℃,反应90 min,PMP与供试品的摩尔比为12∶ 1,NaOH与供试品的摩尔比为2∶ 1;色谱条件为:0.1 mol/L 磷酸盐(pH 6.7)缓冲液-乙腈(82∶ 18,V/V),检测波长:245 nm,流速:1 mL/min.在此色谱条件下,甘露糖醛酸(M)和古罗糖醛酸(G) 分离良好,测得PSS、PMS、PGS的M/G分别为2.37±0.05、6.60±0 22和0.22±0 03,该方法具有良好的精密度和重现性,灵敏度高,适合于海洋褐藻多糖类药物的微量分析.     

人参多糖部分酸水解物的HPLC-ESI-QTOF-MS分析

本文采用酸水解方式,获得人参多糖的部分水解产物,通过高效液相色谱-电喷雾电离高分辨飞行时间质谱(HPLC-ESI-QTOF-MS)对人参多糖部分酸水解产物进行分析,建立人参多糖糖谱研究的方法。同时结合几种标准二糖的MS和MS/MS分析,建立依靠质谱分析确定多糖糖苷键类型的方法。研究结果确定了5种糖苷键链接类型在MS/MS中的断裂规律,并发现人参多糖的糖苷键链接类型为1,3糖苷键和1,4糖苷键。本文研究方法,具有简单快速、稳定性好、准确度高等优点,为中草药的指纹性糖谱研究提供了新的借鉴方法。     

糖醛酸的气相色谱分析

糖醛酸是许多具有生物活性的多糖的基本组分之一,广泛存在于动植物和微生物中。其分离和鉴定有比色、脱羧、纸上电泳、纸色谱、离子交换等方法。糖醛酸的气相色谱分析是糖的气相色谱分析方法的发展。与以上方法相比,具有灵敏、准确、糖醛酸分得开等特点,早为人们所重视。早期的有Perry等的工作,是将糖醛酸还原为糖醇酸,再转化为糖醇酸内酯,然     

塑料食品包装中对苯二酸向食品模拟物迁移的高效液相色谱测定方法

建立了高效液相色谱(HPLC)法测定塑料食品包装中对苯二酸(TA)向食品模拟物的迁移量.对前处理净化技术及色谱条件进行了优化,并利用超高效液相色谱-串联质联(UPLC-MS/MS)进行确证.该方法在0.3～30 mg/L范围内线性关系良好,检出限为0.3 mg/L.食品模拟物中对苯二酸的加标回收率在89.0%～110.0%之间,其相对标准偏差为0.45%～9.24%,重复性和再现性良好.利用该方法对实际塑料包装制品中对苯二酸进行了迁移测定.     

氟化衍生-固相萃取对水中痕量膦酸的GC-MS检测EI北大核心CSCD

针对水中痕量(<1 mg/L)甲基膦酸类化合物进行了GC-MS定性分析检测研究。建立了固相萃取结合氟化物衍生的方法进行样品制备。采用600MHz核磁对氟化物衍生效率进行分析,衍生效率大于99%。采用气相色谱-电子轰击电离质谱(GC-EI/MS)、气相色谱-化学源负电离质谱(GC-NCI/MS)以及气相色谱-选择离子扫描质谱(GC-SIM-MS)分析方法对5种标准物质沙林、梭曼原体以及甲基膦酸异丙酯、甲基膦酸乙酯、甲基膦酸的衍生化产物进行了分析,检出限分别为10,50,0.2,0.1,0.05μg/L,方法的相对标准偏差小于7%。     

一种海洋弧菌(Vibrio sp. WYA)褐藻胶裂合酶降解特性研究

从海洋弧菌(Vibrio sp.  WYA)中得到一种褐藻胶裂合酶， 将其分别作用于寡聚甘露糖醛酸和古罗糖醛酸纯品(dp5~7)以及褐藻胶， 采用HPTLC和FPLC等技术对产物进行分析， 并应用ESI\|MS和NMR进行结构分析. 结果表明， 该酶最小识别片段为六糖， 终产物主要为三糖， 且识别和切割位点为甘露糖醛酸残基.     

固相酸解法制备古糖酯寡糖及其电喷雾质谱分析

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

氟化衍生-固相萃取对水中痕量膦酸的GC-MS检测

针对水中痕量(1 mg/L)甲基膦酸类化合物进行了GC-MS定性分析检测研究。建立了固相萃取结合氟化物衍生的方法进行样品制备。采用600MHz核磁对氟化物衍生效率进行分析,衍生效率大于99%。采用气相色谱-电子轰击电离质谱(GC-EI/MS)、气相色谱-化学源负电离质谱(GC-NCI/MS)以及气相色谱-选择离子扫描质谱(GC-SIM-MS)分析方法对5种标准物质沙林、梭曼原体以及甲基膦酸异丙酯、甲基膦酸乙酯、甲基膦酸的衍生化产物进行了分析,检出限分别为10,50,0.2,0.1,0.05μg/L,方法的相对标准偏差小于7%。     

阿卡波糖对Ⅱ型糖尿病大鼠尿液代谢轮廓的影响

采用超高效液相色谱-质谱联用(UPLC-MS/MS)方法研究了阿卡波糖对Ⅱ型糖尿病大鼠代谢轮廓的影响, 分析了健康组、 糖尿病模型组和糖尿病给予阿卡波糖组的大鼠尿样, 采用主成分分析法(PCA)和偏最小二乘法-判别分析(PLS-DA)对数据进行分析. PCA得分图表明, 健康组、 糖尿病组和阿卡波糖组的代谢轮廓有显著差别, 根据PLS-DA载荷图筛选, 将对各组分离贡献大的化合物的串联质谱分析数据经Human Metabolome Database(HMDB)和Mass Bank.jp等数据库检索, 进行质谱信息匹配, 鉴定出苯乙酰甘氨酸、 肌酐及葡萄糖酸等8种内源性代谢物为潜在生物标记物.     

Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer

Matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) was applied to characterize permethylated oligosaccharides. Under these ionization conditions such derivatives yield intense signals corresponding to sodium-cationized molecular species. A systematic study was conducted on a series of neutral and sialylated permethylated oligosaccharides to allow rationalization of the fragmentation processes. The major fragments observed in the MALDI-TOF/TOF-MS/MS spectra result from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine and sialic acid residues. The fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting these oligosaccharides, and 'internal' cleavage ions which are derived from elimination of substituents from around the pyranose ring, were also observed. This extensive fragmentation was shown to be useful for the structural characterization of oligosaccharides. MALDI-TOF/TOF-MS/MS of permethylated oligosaccharides appears to be a powerful tool for carbohydrate structural analysis.     

杂合褐藻糖胶寡糖的制备及结构分析

采用热水提取法从海蒿子(Sargassum pallidum)中得到一个杂合的褐藻糖胶(SPF); 采用稀酸水解和低压凝胶渗透色谱(LPGPC)分离得到一系列杂合硫酸寡糖. 结合单糖组成、 甲基化和电喷雾碰撞诱导串联质谱(ES-CID-MS/MS)分析表明, 所得21个寡糖属于杂化岩藻寡糖硫酸酯, 主要由α1→3连接的Fuc及少量β1→4连接的Xyl和β1→6连接的Gal组成; 硫酸基取代位点主要存在于Fuc的C4或C2位、 Xyl的C2位和Gal的C4位; Fuc主要存在于寡糖的非还原端. 实验结果表明, ES-CID-MS/MS 技术可用于各种杂合褐藻糖胶寡糖的结构序列分析. 这些结构多样的硫酸寡糖可进一步点印到糖芯片上, 研究其与蛋白相互作用.     

Systematic identification of N-acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation

Recently, a useful procedure for the preparation of both even- and odd-numbered series of N-acetylheparosan (NAH) oligosaccharides was established. The present report describes findings when these NAH oligosaccharides were subjected to comparative mass spectrometry (MS)/MS fragmentation analysis by matrix-assisted laser desorption/ionization (MALDI)-LIFT-time-of-flight (TOF)/TOF-MS/MS, and electrospray ionization (ESI) collision-induced dissociation (CID) MS/MS. The resultant fragment ions were systematically assigned to elucidate fragmentation characteristics. In the MALDI-LIFT-MS/MS experiments, all the NAH oligosaccharides underwent unique glycosidic cleavages that included B-Y ion cleavages (nomenclature system of Domon and Costello, Glycoconjugate J. 1988; 5: 397) at the C-1 side, and C-Z ion cleavages at the C-4 side, with respect to glucuronic acid (GlcA). In addition, (0,2)A and/or (0,2)X cross-ring cleavages were observed for relatively small oligosaccharides. The former observation clearly reflects the occurrence of a GlcA-N-acetylglucosamine (GlcNAc) alternating structure of NAH, while the latter feature implies the occurrence of the -beta-1-4-glucuronide linkage. Extensive glycosidic cleavages were also observed in the ESI-CID-MS/MS fragmentation, though cleavage specificity was less evident than in the case of MALDI-LIFT-TOF/TOF-MS/MS. The information obtained in this study should be valuable for understanding both biosynthetic and degradation processes of NAH and its derivatives including heparin and heparan sulfate, as well as artificially modified NAH oligosaccharides.     

Structural characterisation of underivatised olive pulp xylo-oligosaccharides by mass spectrometry using matrix-assisted laser desorption/ionisation and electrospray ionisation

Purified olive pulp glucuronoxylans, with a Xyl/GlcA ratio of 7:1, were subjected to mild acid hydrolysis and the mixture of oligosaccharides obtained was fractionated by size exclusion chromatography. One elution fraction representative of low molecular weight oligosaccharides was analysed by mass spectrometry using matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) as ionisation methods, in the positive mode. Both types of spectra showed cationised molecules [M + Na](+) of xylo-oligosaccharides in a range below m/z 1,000. The xylo-oligosaccharide structures identified were series of neutral oligosaccharides of xylose (Xyl(n), n = 3-7), of acidic oligosaccharides substituted by one glucuronic acid (Xyl(n)GlcA, n = 3-5) and by two glucuronic acid residues (Xyl(n)GlcA(2), n = 2 and 3), and also of acidic oligosaccharides substituted with one 4-O-methylglucuronic acid residue (Xyl(n)meGlcA, n = 2-4). The proposed structures were confirmed by tandem mass (MS/MS) spectra obtained using collision induced dissociation of the molecular ions. Fragmentation of cationised adducts of neutral Xyl(n) yielded C- and A-type fragments, while ammonium adducts mainly yielded B-type fragments. The fragmentation of the sodium adducts of acidic oligosaccharides (Xyl(n)meGlcA, Xyl(n)GlcA) resulted in the loss of the substituting residue (GlcA or meGlcA) as the predominant fragment, while the corresponding ammonium adducts yielded B-type fragments.     

Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis

Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).     

HPLC/RI与HPLC/ESI-MS方法研究细菌D-97酶合成海藻糖的过程

 通过高效液相 /示差折光检测系统 (HPLC/RI)分析可获得细菌D 97利用糊精或淀粉水解物合成海藻糖的基本生物学信息 ,包括微生物培养碳源对细菌D 97胞内海藻糖合成酶系的影响以及该酶系利用不同种类或不同分子链长度的麦芽寡糖合成海藻糖的能力及作用过程。采用HPLC与RI及电喷雾电离质谱 (ESI MS)联用并结合其他生物学手段对由细菌D 97获得的纯酶组分 (酶A)作用产物进行定量和定性分析 ,从而基本明确了D 97胞内酶合成海藻糖的过程。     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

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

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

Structural characterization of 2-aminobenzamide-derivatized oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight tandem mass spectrometer

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.     

N-linked oligosaccharide analysis of rat brain Thy-1 by liquid chromatography with graphitized carbon column/ion trap-Fourier transform ion cyclotron resonance mass spectrometry in positive and negative ion modes

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.