CHARACTERIZATION OF SULFATIDES AND GAGS IN HUMAN MILK

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 糖缀合物和寡糖，含有特定碳水化合物结构的分子，构成抑制病原体的主要类别的乳组分。我们对鞘糖脂特别感兴趣，鞘糖脂由具有N-连接脂肪酰基的寡糖鞘氨醇碱组成。最近的发现表明，  聚糖上带有硫酸酯基的鞘糖脂  可能在抗击艾滋病方面发挥重要作用。然而，只有来自人乳的硫苷脂，而不是来自其他哺乳动物来源的硫苷脂，例如，牛脑有这种生物活性。我们开发了从人乳中提取鞘糖脂和糖胺聚糖的方法，并用LesTQ Orbitrap MS研究了它们的结构。 将合并的人母乳样品冻干、复溶并通过Folch分配。将级分依次干燥和萃取，并用柠檬酸柱分离。硫酸脂标准品用作对照。结构表征基于在LTQ-Orbitrap质谱仪上以负离子模式与Triversa Nanomate系统偶联进行的串联MS分析。在设计的工作流程下，对信号强度最高的前体离子分别采用CID和HCD作为碰撞方法。 从使用LTQ-Orbitrap获得的负离子ESI MS光谱中，检测到许多酸性鞘糖脂。HCD用于观察较低质量范围的碎片，而CID用于补偿HCD在较高质量范围内的较低效率。通过结合这些技术，我们能够在低丰度和高丰度峰的范围内分配聚糖部分和神经酰胺结构。脂肪酰基链长从16到24个主链碳不等，具有不同程度的不饱和度。由于具有硫酸根或磷酸根基团的GSL的确切质量非常接近，因此不可能在碎片化之前分离出具有单一组成的离子，但MS 2光谱中存在诊断产物离子。我们优化了鞘糖脂提取方法，降低了磷脂含量，便于观察硫酸化GSL。通过该LTQ Orbitrap MS工作流程，我们在人乳抗HIV组分中鉴定了数百种脂质。正在进行的实验将进一步揭示人乳GSL和GAG组分中的化合物结构，并测试合成硫苷脂抗HIV的活性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycoconjugates and oligosaccharides, molecules containing specific carbohydrate structures, constitute a major class of milk components that inhibit pathogens. We are particularly interested in glycosphingolipids, which consist of an oligosaccharide sphingosine base with an N-linked fatty acyl group. Recent discoveries have indicated that sulfatides  glycosphingolipids carrying a sulfate ester group on the glycan  may play an essential role in fighting HIV. However, only sulfatides from human milk, not those from other mammalian sources, e.g., bovine brain, have this bio-activity. We have developed approach to extract glycosphingolipids and glycosaminoglycans out of human milk and study their structures by LesTQ Orbitrap MS. The pooled human breast milk samples were lyophilized, reconstituted and put through Folch partition. The fractions were dried and extracted in turn and separated with a silicic acid column. Sulfatide standards were used as a control. Structural characterization was based on tandem MS analyses performed on an LTQ-Orbitrap mass spectrometer coupled with a Triversa Nanomate system in the negative ion mode. Under the designed work flow, CID and HCD were applied as collision methods to the precursor ion which had the highest signal intensity. From the negative-ion ESI MS spectra obtained with the LTQ-Orbitrap, numerous acidic glycosphingolipids were detected. HCD was used for observing the lower mass range fragments, while CID was applied to compensate for the lower efficiency of HCD in the higher mass range. By combining these techniques, we were able to assign both the glycan moiety and the ceramide structures across the range of low and high abundance peaks. The fatty acyl chain lengths varied from 16 to 24 backbone carbons with different degrees of unsaturation. Since the exact masses of GSLs with sulfate or phosphate groups are very close, it was impossible to isolate ions having a single composition prior to fragmentation, but diagnostic product ions were present in the MS2 spectra. The glycosphingolipid extraction method we optimized reduced content of phospholipids and facilitated observation of the sulfated GSLs. By this LTQ Orbitrap MS work flow, we identified hundreds of lipids in the human milk anti-HIV fraction. Ongoing experiments should reveal further compound structures in the human milk GSL and GAG fractions and test the activity of synthetic sulfatides against HIV.