CHARACTERIZATION OF SULFATIDES AND OTHER LIPID CONJUGATES IN HUMAN MILK

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。 鞘糖脂(GSLS)和磷脂(PLS)是细胞膜的组成部分，调节其生物物理性质，参与多种生物学过程。人乳硫脂，而不是来自牛脑的那些，对人类巨噬细胞和淋巴细胞的HIV感染是有效的。生物学作用依赖于特定的寡糖和神经酰胺部分。磷脂酰肌醇的极性基团可由乙醇胺、胆碱、丝氨酸、肌醇或磷脂酰甘油组成。酰基的饱和/羟化程度和脂肪酰化链可能不同。我们正在使用振动冷却的MALDI-FTMS来检测TLC分离的物种，然后通过Sori-CAD和IRMPD来有效地裂解它们，以确定结构细节。用氯仿/甲醇/冰醋酸/水(65；25；3；1v/v)薄层层析分离脂类标准品或从母乳中提取的脂类；然后将饱和基质溶液喷雾到靶标上，并用VC-MALDI-FTMS扫描。使用氮气碰撞气体和IRMPD技术进行Sori-CAD碎裂。脂类直接从薄层色谱板表面MALDI解吸，并用冷却气体加热。用SWIFT分离感兴趣的峰，用Sori-CAD和IRMPD将其碎裂。在每个扫描步骤之后，发现了大量中性和酸性的GSL和PL同系物。较短的IRMPD脉冲定义了糖基，高能辐射建立了神经酰胺结构。将SOI-CAD和IRMPD应用于硫脂，得到了具有HSO4-和己糖硫酸盐特征的高丰度峰。脂肪酰链长度不同(C16-C24)；有些是羟化的。从一个TLC斑点上解吸的四个主要物种产生的(+)模谱显示两个油酰基的顺序消除；从这些斑点获得的(-)模质谱显示C16：0、C18：0、C18：1和C18：2酰基链的丢失。用Sori-CAD对两个Rf值较低的斑点进行分析，结果表明存在棕榈酰基和油酰基(34：1)或棕榈酰基和亚油酰基(34：2)取代的甘油磷脂，而Rf值较高的两个点存在亚油酰基和硬脂酰基(36：2)和亚油酰基和油酰基(36：3)取代基。其他不寻常的磷脂酰衍生物也出现了，并正在进一步研究中。目前的研究还使用MALDI-TOF MS对Bruker Reflex IV和LC/MS/MS对Q-Star和LTQ-Orbitrap MS进行糖脂结构的归属。

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 and LTQ-Orbitrap MS to assign glycolipid structures.