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.
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