MASS SPECTRAL ANALYSIS OF NOVEL BACTERIAL LIPOPOLYSACCHARIDES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 脂多糖（LPS）及其脂质A（LA）锚是已知的内毒素，因为它们在感染性疾病的起源中起重要作用。海洋细菌LPS参与了其自身的环境适应过程。我们正在调查是否从海洋细菌的LA的低内毒素可能会导致显着的差异，在其结构相比，致病同源物。还预计，海洋脂质A结构的不寻常和未知的模式的确定，可以促进改进的协议的脂质A的致病菌株的结构表征。本研究以假交替单胞菌属和海洋单胞菌属的两株菌为例，采用质谱解离和化学降解相结合的方法。通过在乙酸盐缓冲液中温和的酸性水解，将处理过的悬浮液冷却和离心，并用氯仿萃取，从LPS悬浮液中获得粗LA。使用更苛刻的酸处理以获得部分脱-O-酰化的LA梯形。采用薄层色谱法（PLC）分离磷酸酰化类型，然后进行质谱分析。用于解离气态阳离子和阴离子LA物质的MS方法包括以下：ESI ITMSn; PSD MALDI-TOF MS和NSCD ESI-、SORI CAD VC-MALDI-和ESI IRMPD-FTMS以及NMR。利用这些方法获得了确定海洋脂质A分子结构的数据。M.发现vaga和P. haloacetontis菌株在共同的脂质A骨架上分别具有单-和二-磷酸/五-酰基同质模式。然而，所获得的ESI和MALDI光谱的配置文件是非常复杂的，特别是在假交替单胞菌属的菌株的情况下。最后一个属的5个以上的物种进行了筛选的MS方法。两个单一的同质初始脂质A型样品，这是回收的假交替单胞菌属，在配置文件中表示的离子簇，延伸超过80 Da的范围。然而，脂质A分子携带只有四个脂肪酸[OH 10：0; OH 11：0; OH 12：0; OH 13：0]的5个位置。这种模式可以包括至少64种分子类型，这些分子类型由脂肪酸池中的前体而不是单一酸产生。MS分析表明，粗脂质A的磷酸/酰基异质性的结果从LPS分离过程中的化学处理。随后的实验探索了使用电子捕获解离和LC/MS/MS表征脂质A。只要细菌不突然遇到新的环境，初始脂质A在体内是相当均匀的。在M菌株的LPS中发现了突变型脂质A。vaga细胞首次转移到改良培养基中时，在COBASE为前苏联和美国科学家交流提供的赠款的支持下，BUSM资源与俄罗斯科学院远东分支之间的合作始于Yelkine博士对BUSM的访问。 Costello教授后来访问了海参崴，Yelkine博士返回后加入了资源人员，继续进行这项调查。随着数据解释的进行和几份手稿的编写，该项目仍在继续。该项目的摘要在2009年在圣胡安举行的国际糖缀合物会议上发表。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lipopolysaccharides (LPS) and their lipid A (LA) anchors are known endotoxins due to their important role in the origin of infectious diseases. Marine bacterial LPS are involved in their own environment adaptation processes. We are investigating whether the low endotoxicity of LAs from marine bacteria may result from remarkable differences in their structures, compared to those of pathogenic congeners. It is also expected that determination of unusual and unknown patterns of marine lipid A structures could facilitate improvements in protocol for structural characterization of lipids A of pathogenic stains. In this study, a combination of mass spectrometric dissociation and chemical degradation techniques are used, taking as the examples two strains from the Pseudoalteromonas and Marinamonas genus. Crude LAs were obtained from LPS suspension by gentle acidic hydrolysis in acetate buffer, cooling and centrifugation of treated suspension, and extraction with chloroform. More harsh acidic treatment was used to obtain the partially de-O-acylated LA ladder. Preparative layer chromatography (PLC) was used isolate phosphate-acylated types and was followed mass spectral analysis. MS methods for dissociation of gaseous cationic and anioinic LA species included the following: ESI ITMSn; PSD MALDI-TOF MS and NSCD ESI-, SORI CAD VC-MALDI- and ESI IRMPD- FTMS, as well as NMR. These methods were utilized to obtain data for determination of the molecular structures marine lipids A. The lipids A of M. vaga and P. haloplanktis strains were found to have mono- and di-phosphate/ penta-acyl homogeneous pattern, respectively, on a common lipid A backbone. However, the profiles of the obtained ESI and MALDI spectra were very complex, especially in the case of the strains of the Pseudoalteromonas genus. More than 5 species of the last genus were screened by MS methods. Two single homogeneous initial lipid A type samples, which were recovered from the Pseudoalteromonas genus, were represented in the profiles by ion clusters that extended over a range of 80 Da. However, lipid A molecules carried only four fatty acids [OH10:0; OH11:0; OH12:0; OH13:0] on five positions. Such a pattern may include at least 64 molecular types arising from precursors in a fatty acid pool instead of a single acid. MS analyses indicated that the phosphate/acyl heterogeneity of crude lipids A results from chemical treatments during the isolation from LPS. Subsequent experiments have explored the use of electron capture dissociation and LC/MS/MS for characterization of lipid A. The initial lipid A is rather homogeneous in vivo so long as the bacteria do not suddenly encounter a new environment. Mutant lipids A were found in the LPS of strain M. vaga when its cells were transferred to the modified medium for the first time. With support from a COBASE grant for exchange of scientists from the former Soviet Union and the US, this collaboration between the BUSM Resource and the Far East Branch of the Russian Academy of Sciences was initiated with a visit of Dr. Yelkine to BUSM. Prof. Costello later visited Vladivostok and Dr. Yelkine returned to join the Resource staff to pursue this investigation. The project continues as data interpretation is carried out and several manuscripts are being prepared. A summary of the project was presented in a lecture at the 2009 International Glycoconjugates Meeting in San Juan, PR.