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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 虽然纳米质谱仪是表征简单脂类混合物(1，2)的一个很好的选择，但对于高度复杂的样品的定性和定量分析往往是不够的。所描述的大多数分离方法都是有限的，因为它们要么只针对感兴趣的特定类别(3)，要么不适合于MS，这是一种更好的检测方法，特别是对于少量样品的分析。我们开发了一种简单、可重现的三步法，通过采用文献中描述的分离系统来进行脂类分析(4，5)。经过可选的初始分级后，正相高效液相色谱-质谱仪首先提供分类分离，然后反相LC-MS/MS系统回答剩余的问题。 方法：(1)分离提取低密度脂蛋白和脂类标准品，用一维或二维LC分离，以正、负离子模式进行质谱分析。使用两种不同的梯度来分离更非极性的和更极性的脂类。量化是基于这一步的。(C)可使用三重四极杆、QoTOF MS或LTQ-Orbitrap MS，或通过纳米级MS/MS和/或前驱体离子扫描，进一步用LC-MS/MS对得到的馏分进行表征。 含有不同非极性、磷脂和糖脂的脂类和糖脂标准品已根据极性被重复分离。当用于生物样品时，这一步骤也用于保护下面的柱，但并不总是必要的。量化的准确性在很大程度上取决于可用的内部和外部标准的质量。 收集的馏分用纳米级联用质谱(MS/MS、前驱体离子扫描和中性损失扫描)进行部分研究，以确定存在的分子物种。在反相柱上可以实现分子物种的干净分离，特别是对于低丰度的PE。 Lcms方法提供了相当健壮且技术简单的方法 复杂脂类混合物的研究。我们已经将这些方法应用于与全长和截短载脂蛋白相关的脂类的分析，在正常个体和具有导致截短蛋白产生的基因修饰的个体中，以及其他生物来源的脂类和糖脂。我们已经公布了低密度脂蛋白的结果，并引起了极大的兴趣，从来自美国和其他地方的研究人员的数量来看，他们在开始在自己的实验室实施这种方法时联系了我们。墨菲等人后来发表了对该方法的修改，简化了提取和层析，但保留了磷脂。(8)我们现在正在研究母乳和血脂紊乱患者的血脂。 1)M.Puffer和R.C.Murphy(2003)。质谱学评论22,332-。 2)X.Han.和R.W.Gross(2005)。质谱学版本24,367-412。 3)R.C.墨菲等人。(2001年)。化学。第101版，479-526号。 4)J.Hamilton和K.Comai(1988)。脂23、1046-49和1150-53。 5)W.W.克里斯蒂等人。(1995年)。J.高分辨率。变色剂。18，97-100。 6)F.K.韦尔蒂等人。(1991年)。J·克莱恩。投资。87年，1748-1754年。 7)U·Sommer等人。(2006)J.Lipid Res.47,804-814. 8)P.M.Hutchins等人。(2008)J.Lipid Res.49,804-813。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 have 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). After an optional initial fractionation, normal phase HPLC-MS first provides class separation and then a reversed phase LC-MS/MS system answers remaining questions. Methods: (a) Isolated and extracted LDL lipids and lipid standards are separated by 1D or 2D LCand are detected by mass spectrometry in positive and negative ion modes. Two different gradients are used for the separation of more nonpolar and more polar lipids. Quantification is based on this step. (c) Fractions obtained can be further characterized by LC-MS/MS using a triple quadrupole, 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 collected fractions are partially investigated by nanospray MS (MS/MS, precursor ion scanning and neutral loss scanning) for the determination of the molecular species present. A clean separation of molecular species can be achieved on a reversed phase column, especially with respect to the low abundant PEs. The LCMS methodology provides fairly robust and technically simple methods for the investigation of complex lipid mixtures. We have applied the methods to the analysis of lipids associated with full-length and truncated apolipiprotein in a normal indivicual and one who has a genetic modification that results in production of the truncated protein, and to lipids and glycolipids from other biological sources. We have published the results for LDL (7) and have drawn significant interest, judging from the number of investigators from the US and elsewhere who have contacted us about this approach as they begin to implement it in their own laboratories. Murphy et al.later published a modification to the method that simplifies the extraction and chromatography but leaves behind the phospholipids. (8) We are now investigating lipids from human milk and from persons with lipid disorders. 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. 8) P. M. Hutchins et al. (2008) J. Lipid Res. 49, 804-813.