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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 虽然纳米喷雾MS是表征简单脂质混合物（1,2）的好选择，但对于高度复杂的样品的定性和定量分析通常不够。所描述的大多数分离方法受到限制，因为它们要么仅针对特定的兴趣类（3），要么不适合MS，这是高级检测方法，尤其是对于少量样品的分析。我们以前通过调整文献中描述的脂质色谱法中描述的分离系统，开发了一种简单的，可再现的三步方法，用于脂质分析（4,5,6）。在可选的初始分馏之后，正常相HPLC-MS首先提供了类别的分离，然后逆转了相位的LC-MS/MS系统，回答了剩余的问题。分离和提取的LDL脂质和脂质标准标准通过1D或2D LC分离，并在正和负离子模式下通过质谱法检测。两种不同的梯度用于分离更多的非极性和更多极性脂质。量化是基于此步骤。现在，我们正在简化使用UPLC的方法。分数的特征在于使用ATHE QSTAR QOTOF MS或LTQ-ORBITRAP MS或NANOSPRAY MS/MS和/或前体离子扫描的LC-MS/MS。脂质和糖脂标准含有各种非极性，磷酸和糖脂的标准，已根据极性可重复分离。当用于生物样品时，此步骤也可以保护以下列，但并非总是必要的。量化的准确性主要取决于可用的内部和外部标准的质量。该系统应用于对与王室相关的脂质的分析，以及来自人类牛奶的硫化物级分，这些脂质含量显示出抗HIV的急性。 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）R.C.墨菲等。 （2001）。化学修订版101，479-526。 4）J。Hamilton和K. Comai（1988）。脂质23，1046-49＆1150-53。 5）W.W. Christie等。 （1995）。 J. High Resol。色谱。 18，97-100。 6）F.K。 Welty等。 （1991）。 J. Clin。投资。 87，1748-1754。 7）U。Sommer等。 （2006）J。Lipid Res。 47，804-814。