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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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。我们正在开发一种简单的，可重复的三步法脂质分析，采用文献中描述的脂质色谱分离系统（4，5）。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。 研究方法：（a）将分离和提取的LDL脂质和脂质标准品逐步传递到硅胶60树脂上并用MTBE（甲基叔丁基醚）洗脱，然后用甲醇洗脱。(b)在沃茨/YMC微孔PVA-Sil HPLC色谱柱上进一步分离这两种馏分或完整样品（或标准品组），并在正离子和负离子模式下通过质谱法进行检测。使用两种不同的梯度，一种基于庚烷和MTBE，一种基于在甲酸铵存在下的MTBE和甲醇，分别用于分离更多的非极性和更多的极性脂质。定量基于此步骤。前者需要柱后进料以进行适当的电离。(c)获得的级分可以通过反相LC-MSMS（使用C18 Atlantis毛细管柱，在与三重四极杆或QoTOF MS连接的沃茨CapLC系统上）或通过纳米喷雾MSMS和在任一质谱仪上的前体离子扫描来进一步表征。 脂质标准品含有不同的非极性，磷脂和糖脂已重现分离的极性的基础上，洗脱硅胶60树脂与甲基叔丁基醚和甲醇。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。使用两种不同的梯度在PVA-Sil正相柱上分离样品，一种用于测定非极性脂质，另一种用于测定极性脂质。正相柱上的这些分离允许至少半定量检测。定量的准确性主要取决于可用的内部和外部标准的质量。 通过纳米喷雾MS（MSMS、前体离子扫描和中性损失扫描）对收集的馏分进行部分研究，以确定存在的分子种类。在反相柱上实现了分子物种的清洁分离。特别是低丰度的PE可以通过这种方式得到确认。 LCMS方法为复杂脂质混合物的研究提供了相当稳健且技术简单的方法。我们目前正在努力改进这些结果，我们正在寻找最佳的外部和内部标准进行量化。为了处理更多的样品，自动化和现代仪器保留的脂质分析将是特别有用的。作为第一步，Q-oTOF仪器的数据依赖性采集能力和更高的灵敏度将提供比三重四极杆MS更多的信息，特别是在第二色谱维度之后。理想的脂质工作质谱仪应具有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。

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.