基于离子液体基质的大豆中寡糖成分基质辅助激光解吸电离-质谱成像分析

将离子液体2,5-二羟基苯甲酸丁胺用于改进基质辅助激光解吸电离质谱分析寡糖的定量重复性,并进一步用于大豆和豆叶中寡糖的质谱成像研究.实验中设定正电荷检测模式、激光能量为70%,采用将离子液体2,5-二羟基苯甲酸丁胺甲醇溶液(20%,V/V)直接覆盖样品的简单加基质方法,分析寡糖样品及大豆和豆叶寡糖分布的质谱成像.结果表明,离子液体2,5-二羟基苯甲酸丁胺作为基质,用基质辅助激光解吸电离化质谱分析蔗糖、棉子糖、水苏糖3种寡糖样品,点内重复性RSD<3%,点间重复性RSD<4%,在0.062~1.00 mg/mL 范围内测定的线性相关系数R2≥ 0.996,显示出较好的定量分析潜力.将此基质用于基质辅助激光解吸电离质谱成像分析大豆切片和豆叶表面的二糖、三糖和四糖,得到空间分辨率为150 μm的3种寡糖质谱成像图.3种寡糖在大豆中大致均衡分布,在豆叶的分布均以叶尖为多,并且根据标准曲线和图中的信号强度可以估计其含量.     

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.     

含N-乙酰化肝素寡糖的制备及序列分析

建立了含N-乙酰化肝素寡糖的分离提纯及其序列结构分析方法.首先应用肝素酶Ⅰ深度酶解低分子量肝素来富集含N-乙酰化结构寡糖,通过Bio-Gel P10凝胶色谱法分离制备了包括二糖至十四糖的系列肝素寡糖粗样品,ProPac PA-1强阴离子高效液相色谱(SAX-HPLC)等方法对粗样品进一步分离,提纯得到4种六糖和3种八糖片段.其次应用肝素酶Ⅰ,Ⅱ和Ⅲ复合酶解与HPLC法分析各纯化寡糖的二糖组分,并结合肝素酶Ⅰ底物特异性,初步推断4种六糖和3种八糖的序列结构.在寡糖的糖链两端均含有N-硫酸化二糖,而N-乙酰化二糖分布在糖链当中.应用电喷雾离子阱-飞行时间质谱(ESI-IT-TOF-MS)在负离子模式下进一步表征寡糖并分析其裂解规律.结果表明,各寡糖中均出现大量因SO32-丢失形成的碎片离子峰,六糖中主要有双电荷和三电荷碎片离子峰;在八糖中出现了一系列从双电荷至五电荷的离子峰.各寡糖的双电荷离子峰质荷比进一步确定了上述寡糖的序列结构.六糖的裂解规律表明,裂解主要存在于糖苷键,N-乙酰葡糖胺和糖醛酸上的裂解方式分别为0,2X和0,2Z.本研究提供了切实有效的分离、分析未知结构肝素寡糖序列的新方法.     

液相色谱-电喷雾-四级杆-飞行时间质谱法分析琼胶寡糖

建立超高效液相色谱-电喷雾-四级杆-飞行时间质谱联用技术快速分离鉴定琼胶寡糖的方法.通过分析比较3种色谱柱(BEH Amide、BEH C8及Atlantis T3)对琼胶寡糖的分离结果发现,Amide色谱柱具有最佳优势,在无需样品衍生的状态下,可使聚合度介于3～29的琼胶寡糖得以良好分离,分析迅速,灵敏度高.而衍生后...     

痕量完整天然寡糖链的液态二次离子质谱测定

痕量、未衍生、完整的天然寡糖可用质谱法直接获得重要的结构信息。用负离子LSIMS测出了n mol级IgM中的高甘露糖型和杂交型糖链分子量及序列。对麦芽七糖、异麦芽七糖、古洛糖醛酸十聚体及若干天然寡糖谱图中碎片离子进行了研究,从糖链混合物的质谱图中也能分析出有用的分子结构。     

寡糖衍生化及基质辅助激光解吸电离飞行时间质谱分析方法研究

为提高中性寡糖在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)中的检测灵敏度,建立了以1-(4-氰基苯基)-4-哌啶碳酰肼(CPH)为衍生化试剂对寡糖的标记方法。寡糖的还原端与CPH的酰肼基团反应生成腙,使得寡糖被CPH标记,衍生物以MALDI-TOF质谱进行分析。结果表明:在反应温度95℃,醋酸浓度为0.125%(V/V),CPH过量100倍的条件下,衍生产率可达最大,并且CPH衍生可使中性寡糖在MALDI-TOF质谱中的检测灵敏度提高10倍。本方法简便快速,灵敏度高,适合微量寡糖链的质谱分析。     

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。     

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.     

液相色谱-质谱联用方法用于严重急性呼吸系统综合症冠状病毒结构蛋白质的研究

通过高效液相反相色谱、毛细管反相色谱-串联质谱联用方法对SARS病毒攻击细胞的研究,鉴定出了N、S和M3种SARS结构蛋白质,并准确测定了N蛋白质的分子量。由分子量结果和生物信息学推断的N蛋白质的理论分子量比较,可以确定N蛋白质不存在常见的磷酸化修饰和糖基化修饰或含量很低。进一步的研究表明：N蛋白质可能存在降解现象,其降解机理尚需探索。另外,通过对样品直接酶切后进行两维毛细管分离和串联质谱鉴定与对样品先进行分离,然后再进行毛细管反相分离-串联质谱鉴定方法比较,表明两种方法在蛋白质组学研究中各具优缺点,可根据不同目的选择使用。     

软电离质谱法研究锂系预聚物

高分子化合物的分子量及其分布是研究高分子材料物理性能的最基本参数之一,分子量及其分布的测定在高分子研究与发展中具有极端的重要性.分子量分布不同,聚合物的物化性能不同,所以历来受到聚合工作者的重视,传统的分析方法是利用凝胶渗透色谱法(GPC),该方法自从本世纪60年代问世以来,使得高分子化合物的分子量及分子量分布的测定变得快速和可靠.但是该方法的缺点是需要校正样品;测出的是相对分子量,而且仅适合于分子量大于2 000的样品,对于1 000左右的小分子量聚合物则无能为力.本文应用软电离质谱技术FAB-MS、FD-MS、MALDI-TOF-MS对合成的两种锂系预聚物进行了分析,并对分析结果进行了讨论.     

聚丙烯酰胺超高相对分子质量测量不确定度评定

根据SY/T 5862–2008聚丙烯酰胺相对分子质量测量方法,测量了超高分子量聚丙烯酰胺的相对分子质量。经过对聚丙烯酰胺相对分子质量测量过程引入的不确定度进行分析和评定,不确定度的来源主要是试样目标液配制过程中引入的不确定度,其次是相对分子质量测量重复性、试样固含量的测定以及温度所引入的不确定度。聚丙烯酰胺相对分子质量测定结果为(2500.6±32.6)×104,k=2。     

Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.     

Effect of delay time and grid voltage changes on the average molecular mass of polydisperse polymers and polymeric blends determined by delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The dependence of the calculated average molecular mass of a polyethylene glycol with a large polydispersity on the instrumental parameters adopted in the acquisition of mass spectra using delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (DE MALDI-TOFMS) was investigated. It has been shown that a combined effect of delay times and potential gradients can act on the ion cloud in the source chamber affecting both mass resolution and average molecular mass value of the analyzed polymeric sample. Also examined was a blend of two different polymers (a PEG and a PMMA commercial sample having a similar average molecular mass), which presents an additional problem concerning the discrimination among the different polymer species as a function of the experimental conditions. In this work, the best instrumental conditions to obtain both good resolution and a correct average molecular mass for the examined polydisperse sample are reported.     

Sample exposure effects in matrix-assisted laser desorption—ionization mass spectrometry of large biomolecules

Effects of sample exposure to UV laser irradiation on the matrix-assisted laser desorption—ionization (MALDI) mass spectra of different proteins are reported. The exposure is varied by irradiating the same sample spot with a differing number of UV laser pulses. The ion yield, mass resolution and internal energy content of ejected molecular ions are monitored as a function of the sample exposure. Other parameters that influence the MALDI spectra (and related to sample exposure) such as laser fluence, sample thickness, matrix-to-protein molar ratio, total deposited amount, and molecular mass of the protein, are also examined.     

Concentration effect in hyaluronan analysis by size exclusion chromatography

Summary The concentration effect in size-exclusion chromatographic analysis of hyaluronans of various relative molecular masses (RMM) has been studied. A critical concentration has been found that is negatively dependent on the hyaluronan molecular mass; the higher the biopolymer molecular mass, the smaller the injected sample where the concentration effect should be taken into account for accurate evaluation of molecular mass distribution. At higher temperatures, however, the concentration effect diminishes significantly.     

Number-average molecular mass determination of polymeric material by pyrolysis-gas chromatography

The number-average molecular mass of a polymeric material has been determined by pyrolysis-gas chromatography (Py-GC) via end-group analysis. The major advantage of this technique is that no sample preparation is required. The sample is not required to be in the dilute solution form, and the amount of sample needed is approximately 0.5 mg. Phenyl group-terminated polybutadiene systems have been studied as an example. The application of Py-GC to obtain the end-group concentration, the number-average molecular mass and the limitations of this method are discussed in detail. The success of this development elevates the role of Py-GC as an important technique for end-group analysis for the determination of number-average molecular mass.     

利用物质磁化率和磁导率性质定性分析不同相对分子质量的大分子聚丙烯酰胺

介绍了一项正在研发中的创新分析技术“电磁感应式磁化率化学定性分析方法”的原理和装置结构。采用该技术定性分析不同相对分子质量的大分子-聚丙烯酰胺样品,并对实验数据进行了分析讨论。该技术利用不同单组分样品磁化率、磁导率性质不同的原理,采用电磁感应电动势方法检测样品在磁场中所产生的附加磁场变量强度,以进行定性分析。实验结果说明,磁化率定性分析技术可以分析800万相对分子质量以下的大分子-聚丙烯酰胺样品。     

Elimination of the concentration dependence in mass isotopomer abundance mass spectrometry of methyl palmitate using metastable atom bombardment

An important problem in mass isotopomer abundance mass spectrometry (MIAMS) is the dependence of measured mass isotopomer abundances on sample concentration. We have evaluated the role of ionization energy on mass isotopomer abundance ratios of methyl palmitate as a function of sample concentration. Ionization energy was varied using electron impact ionization (EI) and metastable atom bombardment (MAB). The latter generates a beam of metastable species capable of ionizing analyte molecules by Penning ionization. We observed that ionization of methyl palmitate by EI (70 eV) showed the greatest molecular ion fragmentation and also showed the greatest dependence of relative isotopomer abundance ratios on sample concentration. Ionization using the 3P2 and 3P0 states of metastable krypton (9.92 and 10.56 eV, respectively) resulted in almost no molecular ion fragmentation, and the isotopomer abundances quantified were essentially independent of sample concentration. Ionization using the 3P2 and 3P0 states of metastable argon (11.55 and 11.72 eV, respectively) showed molecular ion fragmentation intermediate between that of EI and MAB(Kr) and showed an isotopomer concentration dependence which was less severe than that observed with EI but more severe than that observed with MAB(Kr). The observed decrease in the dependence of isotopomer abundance on sample concentration with a decrease in molecular ion fragmentation is consistent with the hypothesis that proton transfer from a fragment cation to a neutral molecule is the gas phase reaction mechanism responsible for the concentration dependence. Alternative explanations, e.g., hydrogen abstraction from a neutral molecule to a molecular cation, is not supported by these results. Moreover, the MAB ionization technique shows potential for eliminating one source of error in MIAMS measurements of methyl palmitate, in particular, and of fatty acids methyl esters, in general.     

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.     

Distribution analysis of ultra-high molecular mass poly(ethylene oxide) containing silica particles by size-exclusion chromatography with dual light-scattering and refractometric detection

Two different size-exclusion chromatography (SEC) systems, connected in-line either to a low-angle light scattering (LALS) or to a multiangle light scattering (MALS) detector, are employed for determination of molecular mass distributions (MMD) of poly(ethylene oxide) (PEO) samples having a weight average molecular mass up to eight millions. The detrimental effect of the presence of strongly scattering silica particles in the samples on the light scattering signal can be eliminated using a suitable sample dissolution procedure utilizing silica solubility in aqueous mobile phase. The selection of flow-rate and sample concentration have a large impact on the obtained results. Hydrodynamic retardation phenomena and nonlinearity effects are shown to introduce severe errors in the molecular mass distributions unless flow-rate and sample concentration are kept at sufficiently low levels. Self-compensating ability of the dual detection in flow-rate effects is shown to be the main advantage here. A good agreement between the results obtained using LALS and MALS detection is found provided that a carefully selected angular extrapolation procedure is used in the case of MALS data. Thus, using carefully selected experimental conditions, SEC with light-scattering (LS) and refractometric detection proved to be an efficient technique for MMD characterisation also of ultra-high molecular mass (UHM) PEO polymers.