CHARACTERIZATION OF SULFATIDES AND OTHER LIPID CONJUGATES IN HUMAN MILK: HIV

母乳中硫苷脂和其他脂质结合物的表征：HIV

• 批准号: 7955961
• 负责人: DAVID S. NEWBURG
• 金额: $ 3.01万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955961
• 关键词: Acetic Acids; Biological; Biological Process; Biology; Brain; Cattle; Cell membrane; Ceramides; Characteristics; Chloroform; Choline; Computer Retrieval of Information on Scientific Projects Database; Detection; Ethanolamines; Funding; Gases; Glycerophospholipids; Glycolipids; Glycosphingolipids; Grant; HIV Infections; Hexoses; Homologous Gene; Human; Human Milk; Hydroxylation; Inorganic Sulfates; Inositol; Institution; Length; Lipids; Lymphocyte; Mass Spectrum Analysis; Medicine; Methanol; Oligosaccharides; Phosphatidyl glycerol; Phosphatidylglycerols; Phospholipids; Physiologic pulse; Polysaccharides; Property; Reflex action; Research; Research Personnel; Resources; Role; Scanning; Serine; Solutions; Source; Spottings; Structure; Sulfoglycosphingolipids; Surface; Techniques; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Water; acyl group; interest; irradiation; macrophage

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. Glycosphingolipids (GSLs) and phospholipids (PLs) are components of cell membranes that regulate their biophysical properties and participate in diverse biological processes. Human milk sulfatides, but not those from bovine brain, are active against HIV infection of human macrophages and lymphocytes. The biological roles are dependent on the specific oligosaccharide and the ceramide portions. The polar headgroup of PLs may consist of ethanolamine, choline, serine, inositol, or phosphatidylglycerol. The acyl groups may differ in their degrees of saturation/ hydroxylation and in their fatty acyl chains. We are using vibrationally cooled MALDI-FTMS for the detection of TLC-separated species followed by their efficient fragmentation by SORI-CAD and IRMPD to determine structural details. Lipid standards or lipids extracted from human milk were separated by TLC using chloroform/methanol/acetic acid/water (65;25;3;1 v/v); the plate was then sprayed with the saturated matrix solution, affixed to the target, and scanned by VC-MALDI-FTMS. Fragmentation was performed by SORI-CAD with N2 collision gas and IRMPD techniques. The lipids were MALDI-desorbed directly off TLC plate surfaces and thermalized by the cooling gas. The peaks of interest were isolated by SWIFT and fragmented by SORI-CAD and IRMPD. Numerous neutral and acidic GSL and PL homologs were found following each scanning step. Short IRMPD pulses defined the glycan moiety and higher energy irradiation established the ceramide structures. Application of SORI-CAD and IRMPD to the sulfatides resulted in high-abundance peaks characteristic of HSO4- and hexose sulfate. Fatty acyl chain lengths varied (C16 - C24); some were hydroxylated. Four major species desorbed from a single TLC spot produced (+) mode spectra showing sequential elimination of two oleoyl groups; (-) mode MS obtained from these spots showed evidence for the loss of C16:0, C18:0, C18:1 and C18:2 acyl chains. Analysis of two spots with lower Rf values by SORI-CAD indicated the presence of a glycerophospholipid with palmitoyl and oleoyl (34:1) or palmitoyl and linoleoyl (34:2) substituents, and two spots at high Rf had linoleoyl and stearoyl (36:2) and linoleoyl and oleoyl (36:3) substituents. Other unusual phosphatidyl derivatives were also present and are being further investigated. Current studies also employ MALDI-TOF MS on the Bruker Reflex IV and LC/MS/MS on the Q-Star to assign glycolipid structures.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 鞘糖脂 (GSL) 和磷脂 (PL) 是细胞膜的组成部分，可调节其生物物理特性并参与多种生物过程。人乳硫苷脂（而非牛脑中的硫苷脂）对人类巨噬细胞和淋巴细胞的 HIV 感染具有活性。生物学作用取决于特定的寡糖和神经酰胺部分。 PL 的极性头基可由乙醇胺、胆碱、丝氨酸、肌醇或磷脂酰甘油组成。酰基的饱和/羟基化程度和脂肪酰基链可能不同。 我们使用振动冷却的 MALDI-FTMS 来检测 TLC 分离的物质，然后通过 SORI-CAD 和 IMPD 对其进行有效的破碎，以确定结构细节。使用氯仿/甲醇/乙酸/水(65;25;3;1 v/v)通过TLC分离脂质标准品或从人乳中提取的脂质；然后将板喷洒饱和基质溶液，固定到目标上，并通过 VC-MALDI-FTMS 扫描。 SORI-CAD 使用 N2 碰撞气体和 IMPD 技术进行破碎。脂质直接从 TLC 板表面 MALDI 解吸，并通过冷却气体热化。 通过 SWIFT 分离感兴趣的峰，并通过 SORI-CAD 和 IRMPD 进行片段化。 每个扫描步骤后都发现了许多中性和酸性 GSL 和 PL 同系物。短 IRMPD 脉冲定义了聚糖部分，而更高能量的照射则建立了神经酰胺结构。 SORI-CAD 和 IRMPD 对硫苷脂的应用导致了 HSO4- 和硫酸己糖的高丰度峰特征。脂肪酰基链长度不同（C16 - C24）；有些被羟基化。从单个 TLC 点解吸的四种主要物质产生 (+) 模式光谱，显示两个油酰基的连续消除；从这些点获得的 (-) 模式 MS 显示 C16:0、C18:0、C18:1 和 C18:2 酰基链丢失的证据。 SORI-CAD 对两个 Rf 值较低的点的分析表明存在具有棕榈酰基和油酰基 (34:1) 或棕榈酰基和亚油酰基 (34:2) 取代基的甘油磷脂，而高 Rf 值的两个点具有亚油酰基和硬脂酰基 (36 :2) 以及亚油酰基和油酰基 (36:3) 取代基。 其他不寻常的磷脂酰衍生物也存在并正在进一步研究中。目前的研究还采用 Bruker Reflex IV 上的 MALDI-TOF MS 和 Q-Star 上的 LC/MS/MS 来指定糖脂结构。