Dataset Information

Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation.

ABSTRACT: Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC-MS method that is able to monitor the major lipid classes found in biospecimens (ie. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion HCD fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC-MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC-MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.

SUBMITTER: Bird SS

PROVIDER: S-EPMC3640281 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation.

Bird Susan S SS Marur Vasant R VR Stavrovskaya Irina G IG Kristal Bruce S BS

Metabolomics : Official journal of the Metabolomic Society 20130301 1 Suppl

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondria ...[more]

PMID: 23646040

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Project description:There is a growing need both clinically and experimentally to improve the characterization of blood lipids. A liquid chromatography-mass spectrometry (LC-MS) method, developed for the qualitative and semiquantitative detection of lipids in biological samples and previously validated in mitochondrial samples, was now evaluated for the profiling of serum lipids. Data were acquired using high-resolution, full scan MS and high-energy, collisional dissociation (HCD), all ion fragmentation. The method was designed for efficient separation and detection in both positive and negative ionization mode and evaluated using standards spanning seven lipid classes. Platform performance, related to the identification and characterization of serum triglycerides (TGs), was assessed using extracted ion chromatograms with mass tolerance windows of 5 ppm or less from full scan exact mass measurements determined using SIEVE nondifferential LC-MS analysis software. The platform showed retention time coefficients of variation (CV) of <0.3%, mass accuracy values of <2 ppm error, and peak area CV of <13%, with the majority of that error coming from sample preparation and extraction rather than the LC-MS analysis, and linearity was shown to be over 4 orders of magnitude (r(2) = 0.999) for the standard TG (15:0)(3) spiked into serum. Instrument mass accuracy and precision were critical to the identification of unknown TG species, in part because these parameters enabled us to reduce false positives. In addition to detection and relative quantitation of TGs in serum, TG structures were characterized through the use of alternating HCD scans at different energies to produce diagnostic fragmentations on all ions in the analysis. The lipidomics method was applied to serum samples from 192 rats maintained on diets differing in macronutrient composition. The analysis identified 86 TG species with 81 unique masses that varied over 3.5 orders of magnitude and showed diet-dependency, consistent with TGs linking diet and disease risk.

Project description:Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or ?-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

Dataset Information

Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation.

Publications

Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation.

Similar Datasets

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