CHARACTERIZATION OF SULFATIDES AND GAGS IN HUMAN MILK

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 含有特定碳水化合物结构的糖共轭和低聚糖构成了抑制病原体的牛奶成分的主要类别。我们对鞘糖脂特别感兴趣，它由一个N-连接的脂肪酰基的鞘氨醇低聚糖组成。最近的发现表明，糖链上带有硫酸酯基团的硫脂糖鞘糖脂可能在抗击HIV方面发挥重要作用。然而，只有来自母乳的硫脂具有这种生物活性，而不是来自其他哺乳动物来源的硫脂，例如牛脑。我们开发了从母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。 将混合的人母乳样本进行冷冻干燥、重组并通过Folch分割。各组分依次干燥提取，硅酸柱分离。以硫脂标准品作为对照。结构表征基于在负离子模式下在LTQ-Orbitrap质谱仪和Triversa纳米酸盐系统上进行的串联MS分析。在设计的工作流程下，对信号强度最高的前驱体离子采用CID和HCD两种碰撞方式。 从LTQ-Orbitrap获得的负离子ESI MS谱中，检测到大量的酸性鞘糖脂。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低效率。通过结合这些技术，我们能够指定低丰度峰和高丰度峰范围内的多糖部分和神经酰胺结构。脂肪酰基链长为16~24个主链碳，不饱和程度不同。由于含有硫酸盐或磷酸基团的GSLS的确切质量非常接近，因此不可能在碎裂之前分离出具有单一组成的离子，但在MS2光谱中存在诊断产物离子。对神经鞘糖脂的提取方法进行了优化，降低了磷脂的含量，便于观察硫酸化的神经节苷脂。通过这个LTQ Orbitrap MS工作流程，我们在母乳抗HIV组分中鉴定了数百种脂类。正在进行的实验应该会揭示母乳中GSL和GAG组分的进一步化合物结构，并测试合成硫脂对艾滋病毒的活性。

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.