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

LC-MSN 定性方法

• 批准号: 7601943
• 负责人: Catherine E. Costello
• 金额: $ 1.08万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601943
• 关键词: Biological; Blood capillaries; Chromatography; Class; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Fractionation; Funding; Genetic; Glycolipids; Grant; Heptanes; High Pressure Liquid Chromatography; Institution; Investigation; Ions; Laboratories; Length; Lipids; Literature; Low-Density Lipoproteins; Mass Spectrum Analysis; Methanol; Methodology; Methods; Modification; Molecular; Numbers; Pattern; Phase; Phospholipids; Plant Resins; Production; Proteins; Publishing; Qualitative Methods; Research; Research Personnel; Resources; Sampling; Scanning; Silicon Dioxide; Source; Standards of Weights and Measures; System; United States National Institutes of Health; Water; ammonium formate; base; capillary; feeding; interest; ionization; mass spectrometer; methyl tert-butyl ether

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 have applied the method 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. We have found that the lipid pattern is different in the two cases. these results have recently been published (U Sommer, H Herscowicz, F K Welty and C E Costello, J. Lipid Res, 2006, 47, 804-814) and are drawing 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.. 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 虽然纳喷雾 MS 是表征简单脂质混合物的不错选择 (1,2)，但它通常不足以对高度复杂的样品进行定性和定量分析。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特定的感兴趣类别 (3)，要么不太适合 MS（优越的检测方法），特别是对于少量样品的分析。我们正在开发一种简单、可重复的三步脂质分析方法，方法是采用脂质色谱文献中描述的分离系统 (4,5)。经过可选的初始分级后，正相 HPLC-MS 首先提供类别分离，然后反相 LC-MS/MS 系统回答剩余问题。 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）和甲醇洗脱。 (b) 这两个级分或完整样品（或标准品组）在 Waters/YMC 微孔 PVA-Sil HPLC 柱上进一步分离，并通过正离子和负离子模式的质谱法进行检测。使用两种不同的梯度，一种基于庚烷和 MTBE，一种基于 MTBE 和甲醇（在甲酸铵存在下），分别用于分离更多非极性和更多极性脂质。量化就是基于此步骤。前者需要柱后进料以实现适当的电离。 (c) 获得的级分可以通过使用与三重四极杆或 QoTOF MS 连接的 Waters CapLC 系统上的 C18 Atlantis 毛细管柱进行反相 LC-MSMS 进一步表征，或者通过任一质谱仪上的纳喷雾 MSMS 和前体离子扫描进行表征。 通过使用 MTBE 和甲醇从 Silica 60 树脂中洗脱，可以根据极性可重复地分离含有不同非极性、磷酸和糖脂的脂质标准品。当用于生物样品时，此步骤还可以保护后续色谱柱，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。 收集的级分通过纳喷雾 MS（MSMS、前体离子扫描和中性损失扫描）进行部分研究，以确定存在的分子种类。在反相柱上实现了分子种类的干净分离。特别是低丰度的PE可以通过这种方式得到确认。 LCMS 方法提供了一种相当稳健且技术简单的方法 复杂脂质混合物的研究。 我们已将该方法应用于正常个体和具有导致产生截短蛋白质的基因修饰的个体中与全长和截短载脂蛋白相关的脂质分析。我们发现两种情况下的脂质模式不同。这些结果最近已发表（U Sommer, H Herscowicz, F K Welty 和 C E Costello, J. Lipid Res, 2006, 47, 804-814），并且从美国和其他地方开始在自己的实验室中实施该方法时就该方法与我们联系的研究人员数量来看，这些结果引起了人们的极大兴趣。 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) 遥控墨菲等人。 （2001）。化学。修订版 101, 479-526。 4) J. Hamilton 和 K. Comai (1988)。脂质 23、1046-49 和 1150-53。 5) W.W.克里斯蒂等人。 （1995）。 J.高分辨率。色谱仪18、97-100。 6) F.K.韦尔蒂等人。 （1991）。 J.克林。投资。 87、1748-1754 年。