【作者】 王珍；

【导师】 黄琳娟；

【作者基本信息】 西北大学 ， 细胞生物学， 2011， 硕士

【摘要】 近二十年来,生物质谱技术在糖类物质研究中得到广泛应用,已成为糖链定性定量分析特别是结构解析的强大工具。糖类物质在质谱中的碰撞诱导裂解是进行结构解析的基础,糖链裂解方式及其规律的研究,对糖类物质结构分析具有重要的理论指导意义。为了丰富和发展对糖链裂解规律的认识,为糖链结构解析提供参考依据,本研究分析了几种不同结构、不同连接方式的寡糖衍生物分子离子电喷雾电离多级质谱碎片,得到以下成果和结论：1.寡糖全甲基化及其ESI-MSn的研究采用Ciucanu和Kerek的甲基化方法对七种常见寡糖进行全甲基化,将全甲基化的寡糖产物进行ESI-MSn实验,并对重复产生的多级质谱结果进行比较和结构解析,研究具有不同结构的一些寡糖在多级质谱中的碎裂行为,获得了不同寡糖的一些裂解规律。1)组成不同、连接方式相同的全甲基化二糖多级质谱组成不同、连接方式相同的乳糖Gal-β(1-4)-Glc及麦芽二糖Glc-α(1-4)-Glc的全甲基化二级质谱都产生了m/z 445,m/z 415,m/z 329,m/z 259,m/z 241的碎片峰,其中乳糖m/z 445与m/z 241碎片峰信号强度分别是麦芽糖的2倍。在三级质谱中只有麦芽糖产生了m/z 315(2,4A2断裂)与m/z 372(0,4A2断裂)这两个穿环裂解碎片峰,而乳糖没有产生,并且在三级质谱中麦芽糖的m/z 259的丰度高达90,而乳糖的该峰丰度只有前者的三分之2)组成相同、连接方式不同的全甲基化二糖多级质谱组成相同、连接方式不同的全甲基化蔗糖Glc-α(1-2)- Fru与松二糖Glc-α(1-3)- Fru产生了具有明显区别的二级质谱图谱,蔗糖发生B1或C1断裂产生m/z 259和m/z 241的峰,而松二糖产生了m/z 245的峰,还产生了很高的m/z 431的峰；两种二糖都产生了m/z 359的峰。组成相同、连接方式不同的全甲基化蜜二糖Gal-α(1-6)- Glc与乳糖Gal-β(1-4)-Glc二级质谱中,蜜二糖产生了质荷比相差44的穿环裂解峰：m/z 301与m/z 345、m/z 329与m/z 373,乳糖只产生了m/z 329和m/z 372的穿环裂解。另外,乳糖的二级质谱有B1或C1断裂产生的m/z 241的峰,而蜜二糖中没有；可见1-4连接和1-6连接的二糖二级质谱有很大的区别。本研究所用的几种二糖,例如：麦芽二糖、乳糖、蜜二糖、松二糖、蔗糖都是同分异构体,这些二糖全甲基化后得出了多次重复的具有差别的多级质谱图,可用于这些同分异构体的鉴别。3)含有以上二糖结构的全甲基化三糖和四糖的多级质谱全甲基化棉子三糖Gal-α(1-6)- Glc-α(1-2)- Fru、蔗糖Glc-α(1-2)- Fru.水苏四糖Gal-α(1-6)-Gal-α(1-6)-Glc-α(1-2)- Fru、松二糖(turanose)Glc-α(1-3)- Fru的二级质谱中都产生了m/z 359的峰,而且棉子三糖的三级质谱与水苏四糖的四级质谱中,m/z 359的丰度是100,而本研究中的其它三种糖没有产生该峰,说明产生这种质谱结果是由结构中都含有果糖组分所致。全甲基化水苏四糖四级质谱与棉子三糖三级质谱图相比,棉子糖的三级质谱中除了产生m/z 375的峰以外,其他峰质荷比都与水苏四糖四级质谱峰的质荷比相同。说明质谱数据结果可以用于推断糖链的结构。2.寡糖的紫外及荧光试剂衍生物的ESI-MSn研究糖类物质没有发色基团,为了便于HPLC及LC-MS等分析,经常需要衍生化,使糖类物质带上紫外和荧光基团,带有氨基的芳香族试剂是常用的试剂,衍生之后的糖链与未衍生的糖链相比使质谱灵敏度明显提高。本研究中使用最常见的八种紫外及荧光衍生化试剂对两种同分异构体二糖还原性末端进行标记,得到了纯度很高的标记样品,在ESI-MSn质谱实验中以6-10pmol的量得到很好的信噪比,获得以下结论：1)发现各种试剂标记的二糖在二级及多级质谱中糖苷键断裂主要产生Y型及Z型离子,B型及C型离子不常见；各种试剂标记的二糖多级质谱中,同种试剂标记的L与M的质谱图差别较明显,可见根据该质谱图谱可以鉴定两种同分异构体二糖；研究中无论是糖苷键断裂还是糖环失水所形成的单烯六元环在下一级质谱中都很容易发生穿环裂解,很可能发生了RDA的裂解反应。2)苯胺与对苯二胺标记的两种二糖在多级质谱中裂解行为相似。2-AP与2-氨基蒽醌标记的二糖的碎裂行为相似,这两种试剂标记的糖链比较特殊,在二级质谱中不易发生C-N键的断裂形成失去标记物的峰,该峰的[M+Na+]m/z是349,而其他还原氨化标记的二糖都产生了相对很高的失去标记物峰。3)在该研究中乳糖的去标记物的m/z 349的峰明显比麦芽糖要高,在ABEE、AEC、苯胺、对苯二胺标记的二糖的二级质谱中L的m/z 349峰丰度为100,而相应的M的该峰丰度为20,可见多级质谱中乳糖更容易失去标记物。4)与不饱和糖环相比饱和糖环在多级质谱中不易发生裂解,而2-AP与2-氨基蒽醌标记的二糖比较特殊,其Y1离子在三级质谱中碎片很多；其它试剂标记的Y1离子在三级质谱中碎片很少,几乎没发生裂解,而Z1离子发生了较多的裂解,Z1离子是不饱和糖环。5)PMP标记的二糖多级质谱解析可知,两种二糖组成单体及其异头碳构象不同,产生的多级质谱图谱中,糖苷键断裂后的离子类型不同,丰度的区别更加明显,例如,以m/z 521和m/z 533作为前体离子的三级质谱图谱。我们还发现,与本研究中所用的其它衍生试剂相比,PMP标记的二糖在多级质谱实验中最稳定,例如以m/z 533作为前体离子的三级质谱中产生了丰富的穿环裂解,同样,失去一个PMP分子的以m/z 521为前体离子的三级质谱实验中也产生了很多的离子碎片。更多还原

【Abstract】 Biomass spectrum technology has been widely used in the analysis of carbohydrates during the past two decades, and has become a powerful tool for qualitation, quantitation, and structure investigation of glycans. Studies on glycan fragmentation pattern and law is a significant work for carbohydrate structure elucidation, which is based on collision-induced glycan fragmentation in mass spectrometry. To increase and develope the knowlege about regular fragmentation pattern of glycans and provide essential reference data for their structure analysis, fragments of several oligosaccharide derivatives of different structures and connections in electrospray mass spectrometry were investigated in detail, and the results and conclusions are as follows:1. Oligosaccharides permethylated and analyzed by ESI-MSnCiucanu and Kerek’s permethylation method were employed here to study seven different oligosaccharides, the permethylated oligosaccharides were analyzed by ESI-MSn, and studied the reproducible ESI-MSn results to figure out the law of collision-induced fragmentation.1) ESI-MSn analysis of permethylated glycans with different composition and in the same linkageLactose Gal-β(1-4)-Glc and Maltobiose Glc-α(1-4)-Glc were in the same linkage but with different composition were produced the same fragments by ESI-MS2, m/z 445, m/z415, m/z 329, m/z 259 and m/z 241, in which m/z 445 and m/z241 produced by lactose is twice higher than that of maltobiose. In ESI-MS3, In ESI-MS3, Only Maltobiose produced two different crossing ring cleavage fragments, m/z 315 (2’4A2) and m/z 372 (0,4A2), but lactose did not. The relative abundance of maltobiose m/z 259 was 90, while lactose was only one-third of the former. 2) ESI-MS" analysis of permethylated glycans with different linkage and in the same compositionSignificant differences were also found between permethylated sucrose [Glc-α(1-2)-Fru] and turanose [Glc-α(1-3)-Fru] in MS2 spectrogram, which have the same composition but different linkage. B1 or C1 cleavage of sucrose [Glc-α(1-2)-Fru] occurred, and produced the peaks of m/z 259 and m/z 241, while turanose [Glc-α(1-3)-Fru] generated m/z 245 and a higher peak of m/z 431. But both saccharides generated m/z 359.In MS2 analysis between permethylated melibiose[Gal-α(1-6)-Glc] and lactose[Gal-β(1-4)-Glc], melibiose generated two sets of crossing ring peak(m/z 301 and m/z 345, m/z 329 and m/z 373) which mass difference was 44Da, and lactose produced two crossing ring peak m/z 329 and m/z 372. In addition, lactose occurred B1 or C1 cleavage which only produced crossing ring peak m/z 241, while melibiose was not. So, the linkage of glycans were made a significant difference in MS2 analysis.Several disaccharides used in this study were isomers, such as maltobiose, lactose, melibiose, turanose and sucrose. These disaccharides were permethylated and subjected to MSn analysis reproducibly could be used for the identification of isomers.3) MSn analysis of trisaccharide and tetrasccharide which contain structures like the bisaccharide above.Permethylated raffinose[Gal-α(1-6)-Glc-α(1-2)-Fru], sucrose[Glc-α(1-2)-Fru], stachyose[Gal-α(1-6)-Gal-α(1-6)-Glc-α(1-2)-Fru], turanose[Glc-α(1-3)-Fru] all produced m/z 359 and the relative abundance of m/z 359 was 100 in MS3 analysis of raffinose and MS4 analysis of stachyose, but the others were significantly different, indicating that both raffinose and stachyose were containing fructose.Comparing the difference between MS3 of raffinose and MS4 of stachyose, all peaks they produced were identical except m/z 375 produced in the former one. These results of MS data can be used to infer the structure of glycans.2. ESI-MSn analysis of UV and fluorescent reagents derivatived oligosaccharide Carbohydrates did not have chromophore, in order to facilitate HPLC and LC-MS analysis, it is necessary to derivatize glycans with amino-aromatic reagent to make it bring an UV or fluorescence groups, and these glycan derivatives can be more sensitively detected by mass spectrometry. Eight most commonly used UV and fluorescent derivatizing reagents were employed to analyze disaccharide isomers, derivatized glycan samples of high purity were obtained, and a good signal-to-noise of 6-10pmol in the ESI-MS" mass spectrometry experiments was got.1) The study found that disaccharide labeled by a variety of reagents mainly produced the Y and Z ions in MS" analysis, and B and C ions did not frequently occured. In the multistage mass spectrometry of disaccharide labeled by various reagents, L and M labeled by the same reagents were obviously different, which indicated that the mass spectrum could be identified according to two kinds of disaccharide isomers. We also discovered that pyranosylene ring formed by either glycosidic bond cleavage or dehydration tends to break across the ring, and the cracking reaction RDA may occured.2) Comparative products analysis(MSn) of lactose and maltose derivated by amino benzene and p-phenylene diamine provided a similar spectrum. The 2-AP and 2-aminoanthraquinone derivatives were similar as well, and the cleavage of C-N bond with a [M+Na]+ ion product m/z 349 did not happen easily, while all of the other derivatives by the reductive amination method produced this peak of much high relative abundance.3) In this research the peak m/z 349 formd by C-N bond cleavage from derivatized lactose is obviously high than that of maltose. In MS2 analysis, lactose derivatized by ABEE, AEC, amino benzene and p-phenylene diamine produced the ion m/z 349 with an abundance up to 100, while the abundance of maltose derivatives is just 20.4) Comparing to the unsaturated ring, saturated ring is not easily to cleave in the form of cross-ring. But it is disparate for the 2-AP and 2-aminoanthraquinone derivatives, whose Y1 ion generated several fragments. The Y1 ion of other derivatives generated few fragments, but Z1 produced more cleavage ions, as Z1 ion was unsaturated ring.5) We concluded from the MS" analysis of the disaccharides derivatized by PMP analysis that the ion type generated can be different if there were different monomer composition and anomeric carbon conformation in oligosaccharide, and there could be much more differences in abundance, such as m/z 521 and m/z 533 in the MS3. We also discovered that PMP-labeled disaccharides were most stable in the derivatives studied in the MSn experiment. For example, as a precursor ion m/z 533 produced much more crossing ring cleavage, and PMP-labeled disaccharides is the same.更多还原