荧光辅助糖电泳用于果胶水解产物中寡糖的分析研究

荧光辅助糖电泳(FACE)是首先用荧光衍生化试剂对糖类分子的还原端进行衍生化标记, 然后在一定浓度的聚丙烯酰胺凝胶上进行分离的分析方法, 可同时分析中性糖与酸性糖. 将该方法用于果胶寡糖的分离分析, 对影响FACE的诸多因素如荧光试剂的种类、用量, 荧光衍生化时间、温度, 分离胶浓度及盐、酸等进行了考察, 对果胶寡糖的FACE条件进行了优化, 结果显示: 每1.2 mg无酸且不含盐的果胶寡糖中, 加入0.2 mol/L的ANTS溶液3.75 μL, 1.0 mol/L的NaBH3CN溶液5 μL, 40 ℃衍生化反应16 h后, 在浓度为38%的分离胶上电泳分离, 取得良好的分离效果. 在该实验条件下, 果胶多糖酸水解后得到聚合度为2～16的果胶寡糖混合物, 与质谱分析结果基本一致. 该方法快速、简捷、灵敏、分辨率高, 费用低, 为果胶多糖可控性降解的监测和果胶寡糖的分离分析提供了技术手段.     

毛细管电泳法分析藏红花植物细胞多糖中单糖组成

以对甲氧基苯胺为衍生试剂,采用毛细管电泳法分析了藏红花植物细胞多糖中的单糖组成。对衍生条件进行了优化,并对毛细管分离条件进行了系统的研究。衍生反应在醋酸含量9.5%(v/v)、80 ℃下反应2 h的衍生产率最大,衍生产物紫外检测波长234 nm。在优化的毛细管电泳分离条件(未涂层熔融石英毛细管柱(60 cm(有效长度50 cm)×50 μm),柱温25 ℃,电压20 kV,使用350 mmol/L硼酸电解液(pH 10.21),压力(3.4475 kPa)进样5 s)下,基线分离了11种结构相近的醛糖(来苏糖、木糖、核糖、葡萄糖、甘露糖、半乳糖、鼠李糖、纤维二糖、麦芽糖、乳糖)、酮糖(果糖)的衍生产物。应用该方法定量检测了藏红花植物细胞多糖水解物中糖的成分,各糖的回收率为94.3%～105.4%,相对标准偏差为3.3%～4.6%。     

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的-NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究.     

PMP柱前衍生高效液相色谱法分析杜氏盐藻多糖的单糖组成

建立了柱前PMP衍生高效液相色谱法分离检测8种中性糖、2种糖醛酸及2种糖胺的方法。筛选出适合PMP衍生物分离的色谱柱为Ec lipse XDB-C18,以0.1 mol/L磷酸盐缓冲液-乙腈为流动相,考察了pH值和乙腈体积分数对各种糖的PMP衍生物的保留及分离的影响,确定最佳pH值为6.7,最佳乙腈体积分数为17%;比较了不同衍生反应时间和萃取除杂前不同量盐酸中和反应产物时醛糖-PMP衍生物的峰面积大小变化,从而得到较佳的样品衍生化条件。应用优化的色谱条件和样品制备方法,测定了由杜氏盐藻提取纯化的多糖级分PD4 a和PD4b的单糖组成,其测定结果与高效阴离子交换色谱法的结果基本一致。     

基于MALDI-TOF质谱技术分析人宫颈癌 HeLa细胞N-糖链结构轮廓

以微量HeLa细胞(10⁷个)为对象, 经细胞裂解、还原羧甲基化、胰酶降解和Oasis-HLB柱提取分离得到总糖肽后, 用PNGase F酶解释放N-糖链. 对所得N-糖链用Sep-Pak C₁₈柱纯化后进行完全甲基化衍生, 再采用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)分析HeLa细胞表面N-糖链的结构轮廓. 结果表明, 在获得的34种N-糖链中, 除高甘露糖型、二天线、三天线、四天线和五天线等N-糖链外, 还出现了在某种程度上与肿瘤发生转移相关的特殊平分型和Lewis结构. 利用MALDI-TOF MS技术可快速分析微量癌细胞表面N-糖链的结构轮廓, 为进一步寻找肿瘤糖链标志物及肿瘤的早期预防诊断提供技术支持.     

酸水解-柱前衍生化-HPLC-MS/MS法分析三种即食海产品中的酸性多糖

从扇贝丁、鱿鱼丝和鳕鱼片中提取得到粗多糖,使用1.3 mol/L的三氟乙酸(TFA)进行降解,1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化后,高效液相色谱-三重四级杆质谱(HPLC-MS/MS)分析降解产物中的二糖片段。扇贝丁和鱿鱼丝中的酸性多糖几乎全部为硫酸软骨素;而鳕鱼片不仅含有硫酸软骨素,还含有少量透明质酸,相对含量分别为87.5%和12.5%。     

咪唑类高铼酸盐催化微晶纤维素降解反应研究

以咪唑类高铼酸盐为催化剂,以离子液体1-烯丙基-3-甲基咪唑氯盐([Amim]Cl)为溶剂降解微晶纤维素(MCC)。分别考察反应温度、反应时间、反应物浓度、催化剂用量和结构对纤维素降解反应的影响。结果表明,以5%1-(3-磺酸)丙基-3-甲基咪唑高铼酸盐([mim-(CH_2)_3SO_3H]ReO_4)为催化剂,在微波辅助加热条件下,0.1 g纤维素在2.0 g离子液体[Amim]Cl中于160℃降解30 min,还原糖收率(TRS)和葡萄糖收率最高可达89.6%和46.7%。研究还对咪唑类高铼酸催化纤维素降解反应的催化机理进行讨论,认为催化剂芳环阳离子、ReO-4中Re=O与纤维素分子中羟基的相互作用是促进纤维素降解的关键     

基体辅助激光解吸—电离飞行时间质谱法分析衍生化的寡链糖

以２，５－二羟基苯甲酸（ＤＨＢ）＋间硝基苄醇（ＮＢＡ）混合基体辅助激光解吸电离飞行时间质谱法（ＭＡＬＤＩ）分析甲壳素降解并衍生化的寡链糖，获得了满意的结果。对比其它基体，如：芥子酸（ＳＡ）、α－氰基－４－羟基肉桂酸等（α＿ＣＨＣＡ），２，５＿ＤＨＢ＋ＮＢＡ混合基体解吸电离效果最好：信号强、信噪比高。甲壳素降解产物的质谱分析至今未见报道。     

糖和糖醇的高效液相色谱分析

采用高效液相色谱示差折光检测器（ＲＩ）,对９种糖和五种糖醇进行了分离检测;另以对甲氧基苯胺和苯甲酰氯为衍生试剂,分别对糖和糖醇进行柱前衍生,使其接上紫外吸收基团,然后用紫外检测器（ＵＶ）进行检测。在０．５￣１２ｇ／Ｌ浓度范围内,对糖和糖醇浓度与其衍生物峰面积进行线性回归分析,其相关系数在０．９８４￣０．９９８之间;ＵＶ的检测灵敏度比ＲＩ的要高约１０００倍。     

含磷毒剂及其降解产物的气相色谱-质谱测定方法研究

研究了用台式气相色谱-质谱仪分析测定含磷毒剂及其降解产物,并对降解产物的衍生化方法及它们的色谱行为进行了研究,总结分析了它们的质谱裂解特点及规律,测定了水样中的含磷毒剂及降解产物。     

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.     

HPLC-analyses of plant biomass hydrolysis and fermentation solutions

Summary In this work the analytical separation of biomass hydrolysis and fermentation products is described. Aminobonded phase and ion exchange materials (HPX 87H, HPX 42A, Spherogel Carbohydrate) were used as HPLC stationary phases for the determination of monomeric and oligomeric carbohydrates, carbohydrate degradation and fermentation products. The HPX 42A and the amino-bonded phase columns are especially suitable for the analysis of the gluco oligomers. The HPX 87H column separates the monomeric sugars and their degradation and fermentation products (e.g. furfurals and ethanol) very well. Using the Spherogel Carbohydrate column the lower gluco-oligomers as well as the monomeric sugars and their degradation and fermentation products, can be determined within 35 minutes.     

Determination of lectin in very dilute solution with umbelliferyl sugars by fluorescence quenching

Umbelliferyl sugars have been used to determine the lectin concentration in very dilute solution by fluorescence quenching methods. The Stern-Volmer plot of fluorescence quenching after considering ground-state complexation is obeyed by several sugars. As little as 1 μg of lectin (in 10 μl) and 250 nM of umbelliferyl sugars can be determined. This method offers a simple, non-destructive and rapid method for the determination of lectin activity.     

Application of column liquid chromatography (HPLC) to special problems in food chemistry a laboratory note

Summary Liquid chromatography has been used for the determination of amino acids, sugars and biogenic amines in food. Some special problems, for example, determination of patuline in apple juice, hyoscyamine and scopolamine in french beans preserves, were also solved by HPLC.     

Amperometric Determination of Sugars at Activated Barrel Plating Nickel Electrodes

We report amperometric determination of sugars by using a disposable barrel plating nickel electrode (Ni‐BPE) in this study. The activated Ni‐BPE possesses good reproducibility in flow injection analysis of sugars with a relative standard deviation of 3.16% for 10 consecutive injections of glucose. The electrocatalytic mechanism for the detection of sugars as well as the use as a detector in high‐performance liquid chromatography (HPLC) is investigated. We achieve a good separation of four sugars (glucose, fructose, sucrose, and maltose) in HPLC with favorable sensitivity at a detection potential of +0.55 V vs. Ag/AgCl. The results of wide linear calibration ranges and detection limits in the μM range meet the need of real sample analysis. This detection method is successfully used for quantitative determination of sugars in honey to identify its authentication.     

Deuterium nuclear magnetic resonance spectroscopy and stable carbon isotope ratio analysis/mass spectrometry of certain monofloral honeys

Quantitative deuterium nuclear magnetic resonance spectroscopy (NMR) has been used in conjunction with stable carbon isotope ratio analysis/mass spectrometry to refine the detection of sugars that have been added to monofloral honeys. The 13C content of sugars indicates the type of photosynthetic metabolism of the plant that synthesized them; the deuterium content is more characteristic of secondary metabolism and of environmental factors. Consequently, determination of the 13C content of honeys and of proteins extracted from the honeys can be used to detect the addition of C4 plant sugars (cane or corn), but it does not reveal the addition of C3 plant sugars such as beet sugar. Deuterium NMR gives useful information for some monofloral honeys. NMR measurement is performed on ethanol obtained from fermentation of the honey and extracted by distillation. The isotopic composition of the ethanol indicates the nature of the sugars from which it was derived. Various types of monofloral honeys were studied, and the results obtained with commercially available honeys demonstrate the usefulness of isotopic analysis and the need to compile a database of authentic honeys to validate or affirm certain results.     

Analysis of lignin degradation products by capillary electrophoresis

Summary A method for the quantitative analysis of phenolic lignin degradation products by capillary electrophoresis (CE) with on-column UV detection has been developed. The liquid biomass solutions contain low molecular hemicellulosic sugars and phenolic lignin degradation products with various degrees of polymerization. Special attention has been paid to the monomeric phenolic components of lignin degradation fragments, e.g. derivatives of phenolic acids, aldehydes, and alcohols. Uncoated fused silica capillaries and borate-phosphate buffer systems at moderate pH conditions were used in order to separate the compounds of interest. To provide validation of the method, the same samples were analyzed independently by HPLC using gradient elution on a RP-C18 column. As sugar degradation fragment, furan-2-carboxylic acid was detected.     

Colorimetric determination of furfural and its precursors with azulene; application to air pollution

Two procedures in which azulene is used as the reagent for the determination of furfurals and furfurals and their carbohydrate precursors are described. Alipliatic aldcliydes, alcohols, and 2-deoxy sugars and their derivatives gave weak to negative reactions. The methods have been applied to the estimation of furfurals and furfuralprecursor carbohydrates in effluent from a coffee-roasting industrial plant.     

Automated flow system on-line to LC with postcolumn derivatisation for determination of sugars in carbohydrate-rich foods

Summary A method for the direct determination of carbohydrates in foods using an automated-flow system coupled on-line to a high-performance liquid chromatograph is proposed. The method is based on postcolumn derivatisation of reducing sugars with p-aminobenzoic acid hydrazide in an alkaline medium following elution from the chromatographic column with an acetonitrile: water gradient as mobile phase. The manifold allows non-reducing sugars to be hydrolysed prior to insertion into the chromatograph, thus making them compatible with the derivatisation reaction and with continuous decolorisation of the sample on an activated carbon column. The method allows determination of six sugars in contents between 0.005 and 4% (w/v) with high precision (3.8–5.0%RSD). For analytical validation, it was applied to determination of fructose, sucrose, maltose, lactose and maltotriose in two reference materials (milk powder and sugar). Finally, the method was used to determine sugars in a variety of carbohydrate-rich foods.     

Compound-specific delta13C analysis of individual amino sugars--a tool to quantify timing and amount of soil microbial residue stabilization

There is strong scientific evidence that microbial residues such as amino sugars may be stabilized in soil. However, up to now, no investigation has been carried out to quantify both the amount and timing of such stabilization. This is primarily due to methodological constraints, because it is not possible to differentiate between stabilized (old) and recently produced (new) amino sugars when these biomarkers are conventionally analyzed, e.g. by means of gas chromatography and flame ionization detection. Therefore, the aim of the present study was to test whether compound-specific isotope analysis (delta13C) of amino sugars extracted from soil could be used to differentiate between old and new microbial residues. For this aim a method for the delta13C analysis of individual amino sugars was developed and optimized. First results of delta13C values of glucosamine, galactosamine, mannosamine, and muramic acid in soil samples from two different ecological studies are presented, clearly indicating that discrimination between soil inherent and newly formed amino sugars is possible in stable isotope labeling experiments. Our results further showed that, in the short term (within 1 month), only few amino sugars were built, thus making highly 13C-enriched substrates necessary for the quantification of new amino sugar production and for the determination of amino sugar turnover rates.