MASS SPECTRAL ANALYSIS OF NOVEL BACTERIAL LIPOPOLYSACCHARIDES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脂多糖(LPS)及其脂蛋白A(LA)锚定是已知的内毒素，在感染性疾病的发生中起重要作用。海洋细菌脂多糖参与了自身的环境适应过程。我们正在调查海洋细菌的LAS的低内毒素是否可能是由于它们的结构与致病同类物质的结构显著不同造成的。此外，对不寻常的和未知的海洋类脂A结构模式的确定可能有助于改进致病菌株类脂A的结构表征方案。在本研究中，以假交替单胞菌和海洋单胞菌属的两个菌株为例，采用了质谱解离和化学降解技术相结合的方法。脂多糖悬浮液在醋酸盐缓冲液中温和酸解，处理后的悬浮液冷却、离心，用氯仿提取，得到粗品LAS。采用更为苛刻的酸处理方法得到了部分脱氧酰化的LA梯形图。采用制备层析(PLC)分离磷酰化类型，并进行质谱分析。气态阳离子和阴离子LA物种的MS解离方法包括：ESI ITMSn；PSD MALDI-TOF MS和NSCD ESI-，Sori CAD VC-MALDI-和ESI IRMPD-FTMS，以及核磁共振。利用这些方法获得了测定海洋类脂A的分子结构数据。支原体和盐生浮游支原体的类脂A分别在共同的类脂A骨架上具有单磷酸和二磷酸/五酰基的均一模式。然而，获得的ESI和MALDI图谱非常复杂，特别是假交替单胞菌属的菌株。用MS方法对最后一个属的5种以上植物进行了筛选。从假交替单胞菌属中回收的两个单一均一的初始类脂A型样品，在剖面中由延伸超过80Da的离子团簇表示。然而，脂类A分子只在五个位置携带四种脂肪酸[OH10：0；OH11：0；OH12：0；OH13：0]。这样的模式可能至少包括来自脂肪酸库中的前体的分子类型，而不是单一的酸。MS分析表明，粗脂A的磷脂/酰基的不均一性是从脂多糖分离过程中化学处理的结果。随后的实验探索了使用电子捕获解离和LC/MS/MS来表征类脂A。只要细菌不会突然遇到新的环境，初始的类脂A在体内是相当均匀的。首次将分枝杆菌细胞转入改良培养基时，在其内毒素中发现了突变的脂类A。在COBASE前苏联和美国科学家交流基金的支持下，BUSM资源和俄罗斯科学院远东分院之间的这一合作是在叶尔金博士访问BUSM后启动的。科斯特洛教授后来访问了符拉迪沃斯托克，叶尔金博士回来加入资源工作人员，继续进行这项调查。随着数据解释的开展和几份手稿的编写，该项目仍在继续。

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.