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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。我们正在开发一种简单、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：(A)将分离提取的低密度脂蛋白和脂质标准品逐级传递到硅胶60树脂上，用甲基叔丁基醚(MTBE)洗脱，然后用甲醇洗脱。(B)这两个组分或全部样品(或一套标准)在Waters/YMC微孔PVA-Sil高效液相色谱柱上进一步分离，并以正离子和负离子模式进行质谱分析。使用两种不同的梯度，一种基于正庚烷和MTBE，另一种基于MTBE和在甲酸铵存在下的甲醇，分别用于分离更多的非极性和更多的极性脂肪。量化是基于这一步的。前者需要柱后馈电才能进行适当的电离。(C)可使用Waters CapLC系统上的C18 Atlantis毛细管柱或与三重四极杆或QoTOF MS连接的反相LC-MSMS进一步表征所获得的馏分，或通过纳米喷雾MSMS和任一质谱仪上的前驱体离子扫描来进一步表征。含有多种非极性、磷脂和糖脂的脂类标准品已通过MTBE和甲醇从硅胶60树脂中洗脱，根据极性重复分离。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。样品在PVA-Sil正相柱上使用两种不同的梯度分离，一种用于非极性脂类的测定，另一种用于极性脂类的测定。正常相柱上的这些分离允许至少半定量检测。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(MSMS、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。在反相柱上实现了分子物种的干净分离。特别是低丰度的PES可以通过这种方式得到确认。LC MS方法为研究复杂的脂类混合物提供了一种相当可靠且技术简单的方法。我们目前正在努力改进这些结果，并正在寻找量化的最佳外部和内部标准。为了处理更多的样品，为脂质分析保留的自动化和现代仪器将特别有用。作为第一步，Q-OTOF仪器的数据采集能力和更高的灵敏度将比三重四极杆MS提供更多的信息，特别是在第二维之后。理想的脂类工作质谱计应该具有MSN能力并提供高质量精度，因此较新的IT-MS仪器，特别是FT-MS仪器为这些研究提供了优势。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。

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 are developing 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 passed stepwise onto and eluted off Silica 60 resin with MTBE (methyl t-butyl ether), followed by methanol. (b) Either these two fractions or the full sample (or set of standards) are further separated on a Waters/YMC microbore PVA-Sil HPLC column and are detected by mass spectrometry in positive and negative ion modes. Two different gradients are used, one based on heptane and MTBE, and one based on MTBE and methanol in the presence of ammonium formate, for the separation of more nonpolar and more polar lipids, respectively. Quantification is based on this step. The former requires a postcolumn feed for proper ionization. (c) Fractions obtained can be further characterized by reversed phase LC-MSMS using a C18 Atlantis capillary column on a Waters CapLC system interfaced to either the triple quadrupole or QoTOF MS , or by nanospray MSMS and precursor ion scanning on either mass spectrometer. Lipid standards containing diverse nonpolar, phospho- and glycolipids have been reproducibly separated on the basis of polarity by elution from Silica 60 resin with MTBE and methanol. This step, when used for biological samples, also serves to protect the following column, but is not always necessary. The sample is separated on a PVA-Sil normal phase column using two different gradients, one for determination of nonpolar lipids, and the other for polar lipids. These separations on the normal phase column allow for an at least semi-quantitative detection. 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 (MSMS, precursor ion scanning and neutral loss scanning) for the determination of the molecular species present. A clean separation of molecular species has been achieved on a reversed phase column. Especially the low abundant PEs can be confirmed that way. The LCMS methodology provides a fairly robust and technically simple method for the investigation of complex lipid mixtures. We currently are working on improvements of these results, and we are searching for optimal external and internal standards for quantification. In order to process larger numbers of samples, automation and modern instrumentation reserved for lipid analyses would be particularly useful. As a first step, the data-dependent acquisition capability and higher sensitivity of Q-oTOF instruments will give more information than the triple quadrupole MS, especially after the second chromatographic dimension. The ideal mass spectrometers for lipid work should have MSn capability and provide high mass accuracy, therefore the newer IT-MS instruments and particularly FT-MS instruments offer advantages for these studies. 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.