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

这个子项目是许多研究子项目中的一个 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 鞘糖脂（GSL）和磷脂（PL）是细胞膜的组成部分，调节其生物物理特性并参与多种生物过程。人乳中的硫苷脂（而不是牛脑中的硫苷脂）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的极性头基可以由乙醇胺、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MALDI-FTMS检测TLC分离的物质，然后通过SORI-CAD和IRMPD对其进行有效的片段化，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;然后用饱和基质溶液喷雾平板，固定到靶标上，并通过VC-MALDI-FTMS扫描。 通过SORI-CAD与N2碰撞气体和IRMPD技术进行裂解。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。 在每个扫描步骤之后发现许多中性和酸性GSL和PL同系物。短的IRMPD脉冲定义了聚糖部分，并且更高能量的照射建立了神经酰胺结构。SORI-CAD和IRMPD对硫酸酯的应用产生了HSO 4-和己糖硫酸酯的高丰度峰特征。脂肪酰基链的长度各不相同（C16 - C24）;有些被羟基化。从单个TLC斑点解吸的四种主要物质产生（+）模式光谱，显示两个油酰基基团的顺序消除;从这些斑点获得的（-）模式MS显示C16：0、C18：0、C18：1和C18：2酰基链损失的证据。 通过SORI-CAD分析具有较低Rf值的两个点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，并且在高Rf处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。目前的研究还采用MALDI-TOF MS在布鲁克反射IV和LC/MS/MS在Q-Star分配糖脂结构。

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.