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 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-MS/MS using a C18 Atlantis capillary column on a Waters CapLC system interfaced to the 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 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 (MS/MS, 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 been published (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. recently published a modification to the method that simplifies the extraction and chromatography but leaves behind the phospholipids. (8) 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.
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