LC-MSN METHOD FOR QUALITATIVE & QUANTITATIVE ANALYSIS OF COMPLEX LIPID MIXTURES

LC-MSN 定性方法

• 批准号: 8365492
• 负责人: Catherine E. Costello
• 金额: $ 1.42万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365492
• 关键词: Biological; Biology; Chromatography; Complex; Detection; Fractionation; Funding; Glycolipids; Grant; HIV; High Pressure Liquid Chromatography; Human Milk; Ions; Lipids; Literature; Low-Density Lipoproteins; Mass Spectrum Analysis; Medicine; Methods; National Center for Research Resources; Phase; Phospholipids; Principal Investigator; Prions; Qualitative Methods; Research; Research Infrastructure; Resources; Sampling; Scanning; Source; Sulfoglycosphingolipids; System; United States National Institutes of Health; base; cost; interest

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. While nanospray MS is a good choice for the characterization of simple lipid mixtures (1,2), it is often not sufficient for the qualitative and quantitative analysis of highly complex samples. Most separation methods described are limited, in that they either target only specific classes of interest (3), or are not well suited for MS, the superior detection method, especially for analyses of small amounts of samples. We previously developed a simple, reproducible three-step method for lipid analysis by adapting separation systems described in the literature for the chromatography of lipids (4,5,6). After an optional initial fractionation, normal phase HPLC-MS first provided class separation and then a reversed phase LC-MS/MS system answered remaining questions. Isolated and extracted LDL lipids and lipid standards were separated by 1D or 2D LC and detected by mass spectrometry in positive and negative ion modes. Two different gradients were used for the separation of more nonpolar and more polar lipids. Quantification was based on this step. We are now simplifying this method using UPLC. Fractions are characterized by LC-MS/MS using athe QStar QoTOF MS, or LTQ-Orbitrap MS, or by nanospray MS/MS and/or precursor ion scanning. Lipid and glycolipid standards containing diverse nonpolar, phospho- and glycolipids have been reproducibly separated on the basis of polarity. This step, when used for biological samples, also serves to protect the following column, but is not always necessary. The accuracy of the quantification depends mostly on the quality of internal and external standards available. The system is being applied to the analysis of lipids associated with prions and to sulfatide fractions from human milk that show anti-HIV acrivity. 1) M. Puffer and R.C. Murphy (2003). Mass Spectrometry Reviews 22, 332-64. 2) X. Han and R.W. Gross (2005). Mass Spectrom Rev. 24, 367-412. 3) R.C. Murphy et al. (2001). Chem. Rev. 101, 479-526. 4) J. Hamilton, and K. Comai (1988). Lipids 23, 1046-49 & 1150-53. 5) W.W. Christie et al. (1995). J. High Resol. Chromatogr. 18, 97-100. 6) F.K. Welty et al. (1991). J. Clin. Invest. 87, 1748-1754. 7) U. Sommer et al. (2006) J. Lipid Res. 47, 804-814.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 虽然纳喷雾 MS 是表征简单脂质混合物的不错选择 (1,2)，但它通常不足以对高度复杂的样品进行定性和定量分析。所描述的大多数分离方法都是有限的，因为它们要么仅针对特定的感兴趣类别 (3)，要么不太适合 MS（优越的检测方法），特别是对于少量样品的分析。我们之前通过采用脂质色谱文献中描述的分离系统开发了一种简单、可重复的三步脂质分析方法 (4,5,6)。经过可选的初始分级后，正相 HPLC-MS 首先提供类别分离，然后反相 LC-MS/MS 系统回答剩余问题。分离和提取的 LDL 脂质和脂质标准品通过 1D 或 2D LC 分离，并通过正离子和负离子模式的质谱检测。使用两种不同的梯度来分离更多非极性和更多极性脂质。量化就是基于此步骤。我们现在使用 UPLC 简化该方法。使用 QStar QoTOF MS 或 LTQ-Orbitrap MS 通过 LC-MS/MS 或通过纳喷雾 MS/MS 和/或前体离子扫描对馏分进行表征。含有不同非极性、磷酸和糖脂的脂质和糖脂标准品已根据极性进行可重复分离。当用于生物样品时，此步骤还可以保护后续色谱柱，但并不总是必要的。定量的准确性主要取决于可用的内部和外部标准的质量。该系统正被应用于分析与朊病毒相关的脂质以及具有抗艾滋病毒活性的母乳硫苷脂成分。 1) M. Puffer 和 R.C.墨菲（2003）。质谱评论 22, 332-64。 2) X. Han 和 R.W. Gross (2005)。质谱修订版 24, 367-412。 3) 遥控墨菲等人。 （2001）。化学。修订版 101, 479-526。 4）J.汉密尔顿和K.科迈（1988）。脂质 23、1046-49 和 1150-53。 5) W.W.克里斯蒂等人。 （1995）。 J.高分辨率。色谱仪18、97-100。 6) F.K.韦尔蒂等人。 （1991）。 J.克林.投资。 87、1748-1754 年。 7) U.索默等人。 (2006) J.脂质研究。 47、804-814。