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

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。 虽然nanospray MS是表征简单脂质混合物的良好选择（1，2），但它通常不足以用于高度复杂样品的定性和定量分析。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感兴趣的特定类别（3），要么不太适合MS（上级检测方法），特别是对于少量样品的分析。我们以前开发了一种简单的，可重复的三步法脂质分析，采用文献中描述的分离系统进行脂质色谱分析（4，5，6）。在可选的初始分馏后，正相HPLC-MS首先提供类别分离，然后反相LC-MS/MS系统回答剩余的问题。分离和提取的LDL脂质和脂质标准品通过1D或2D LC分离，并通过质谱法在正离子和负离子模式下检测。两种不同的梯度用于分离更多的非极性和更多的极性脂质。定量基于该步骤。我们现在正在使用UPLC简化此方法。通过LC-MS/MS（使用MQStar QoTOF MS或LTQ-Orbitrap MS）或通过纳米喷雾MS/MS和/或前体离子扫描表征馏分。含有不同非极性、磷脂和糖脂的脂质和糖脂标准品已根据极性进行了可重复分离。当用于生物样品时，该步骤也用于保护随后的柱，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和硫苷脂馏分从人乳，显示抗艾滋病毒的acrivity相关的脂质。 1)M. Puffer和R.C. Murphy（2003年）。Mass Spectrometry Reviews 22，332-64. 2)X. Han和R.W.毛额（2005年）。Mass Spectrom Rev. 24，367-412. 3)R.C. Murphy等人（2001年）。101，479-526。 4)汉密尔顿和K. Comai（1988年）。Lipids 23，1046-49 & 1150-53. 5)W.W. Christie等人（1995年）。高解析度J. High Resol. Chromatogr. 18，97-100。 6)法光Welty等人（1991年）。J. Clin. Invest. 87，1748-1754。 7)联合Sommer等人（2006）J. Lipid Res.47，804-814.

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.