2010全国质谱大会大会报告（二）
台湾国立台湾大学 Guor-Rong Her (何國榮)教授
来自台湾国立台湾大学的Guor-Rong Her (何國榮)教授，做了题为“Comparative studies of Protein Glycosylation Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry”（应用同位素标记的电喷雾离子阱质谱比较研究蛋白的糖基化）的报告。在报告中，何教授介绍道：在糖蛋白分析中，糖蛋白往往在质谱分析前就被胰蛋白酶酶解了。而包括糖蛋白的浓度、糖基化位点、糖苷轮廓（profile）等因素，能够解释当发现特定的糖肽后所产生的信号的差别。课题组发展了一种分析糖蛋白的比较分析方法，能够测定出糖蛋白的浓度、糖基化位点、糖苷轮廓的变化。这种比较分析方法应用了甲醇d0和甲醛d2的同位素标记技术，然后用微晶纤维素富集再进行质谱分析。应用该方法，用核糖核酸酶B进行了对糖蛋白浓度、糖基化位点、糖苷轮廓变化的比较分析实验，并用ESI线性离子阱质谱进行了糖肽的测定。应用仪器具有的MS2和MS3能力，也获得了糖基化位点和糖苷的序列。以下是英文摘要：
In the analysis of glycoprotein, glycoprotein was often digested with trypsin before MS analysis. Several causes including glycoprotein concentration, glycosylation site occupancy and glycan profile may account for the difference if the signal of a specific glycopeptides was found to be different. A strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The change of glycoprotein concentration was studied using samples prepared with different quantity of ribonuclease B. Level of glycosylation site occupancy was studied by mixing different ratios of ribonuclease B and ribonuclease A. The change of the site-specific glycan profile was studied by mixing ribonuclease B treated with α-mannosidase and intact ribonuclease B. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained.
清华大学 张新荣教授
来自清华大学的张新荣教授做了题为“常压解吸附离子源及其应用”的报告。常压解析负离子源（DESI源）是近几年的研究热点。目前的DESI源虽然在兴奋剂快速检测、枪击残留物检测等方面应用广泛，但也存在气体流速大、需要调节的参数多、需要喷雾液、成像分析分辨率低、构造复杂不易小型化的缺点。张教授在报告中提出了介质阻挡放电离子源（DBDI源），与DESI源相比，DBDI源具有没有引入溶剂污染，可以用于气体、液体、固体等样品分析，成本低，成像分析分辨率高且对于被测物损害程度非常小，并且可进行表面分析的优点。张教授介绍，DBDI源直接进行气体分析时比EI源，直接进行液体分析时可进行化学反应的实时过程监测和有机反应的全过程检测，用于固体表面分析时可用于爆炸物分析、阵列分析20种氨基酸、药片的快速分析（将来可能用于质控），在成像分析方面可用于字画鉴定的无损分析等。
张教授总结，DBDI是一种新型的质谱离子化方法，具有适用面广、体积小、成本低的特点，适合作为小型化质谱的离子源；DBDI用于直接分析，无需样品预处理，是一种理想的实时、原位和高通量的分析手段。DBDI能够通过方便调节电压与频率等参数，获得丰富的分子结构信息，甚至元素的信息。利用DBDI的较高的空间分辨能力，能够用于无损（微损）条件下的成像分析。
加拿大国家研究中生物科学研究所 Li, Jianjun（李建军）
来自加拿大国家研究中生物科学研究所的Li, Jianjun（李建军）教授，做了题为“Derivatization strategies for Glycomics”（糖组学的衍生方法）的报告。在报告中，李教授首先介绍了糖组学的基本概念，糖组学的目标是，系统地鉴定和表征不同糖苷的结构、功能、活性和定量等，对发现和鉴定癌细胞、体液、组织中的疾病生物标志物来说，糖组学研究是一个已被公认的创新和综合的方法。李教授随后介绍了课题组在糖组学研究上的新方法。比如特殊的衍生后再进行ESI-MS或MALDI-MS分析。另外，他们还发展了一种糖组学方法，来鉴定和定量复杂生物基体中的目标糖组。并获得了很好的灵敏度和线性动态范围。以下是英文摘要：
Glycomics is the systematic identification and characterization of diverse glycans for their structure, function, activity, and quantity. Global profiling of glycomes of cancer cells, body fluids, or tissues has been considered as an innovative and comprehensive approach to discover and identify biomarkers for various diseases. Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated oligosaccharides in presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrroli-dinophosphonium hexafluorophosphate(PyAOP). After methylamidation, sialylated oligosaccharides can be analyzed by MALDI-MS without loss of sialic acid moiety. The ESI-MS and MALDI-MS analysis of both 3’-6’-sialyllactose derivatives indicated that the quantitatively conversion of sialic acids was achieved, regardless of their linkage types under the optimal derivatization conditions.This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins(Fetuin,human acid glycoprotein and bovine acid glycoprotein)containing different types of complex oligosaccharides.Using this derivatization method,the N-glycans of sera from different species(human, mouse and rat)were successfully characterized by MALDI-MS.Due to the mild reaction conditions,the modification in sialic acid residues can be retained. This improvement makes it possible to detect the sialylated oligosaccharides containing O-acetylated sialic acid moieties using MALDI-MS in positive ion mode.
In the meantime, the development of glycomics is limited by the ability to identify and quantify large numbers of glycans in complex biological samples, which is only partially achieved by current glycomics approaches. I will also present a strategy termed targeted glycomics enabling highly sensitive and consistent identification and quantification of target sets of glycans across multiple samples. The strategy was first applied to the analysis of glycans released from ribonuclease B. A linear dynamic range of three orders of magnitude (0.1-100 ng ribonuclease B ) was achieved for the detection of all five glycans. The method exhibited excellent sensitivity, evident by the detection of limit of 100attomolar for Man9Glc NAc2.The strategy was further demonstrated by characterizing the glycome of pancreas cancer cells. More than 60 N-glycans were identified and detected for about five million cells.
