Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes. Recent developments in mass spectrometric technology enabled the comprehensive characterization of eukaryotic lipidomes, fostering the molecular biology of lipids and metabolism-related disorders (reviewed in Refs. 1-4). Typically, lipidome profiling by mass spectrometry proceeds along LC-MS or shotgun approaches. The former identifies and quantifies lipid species preseparated by normal or reversed-phase chromatography coupled online to a mass spectrometer, which is capable of fast acquisition of MS or MS/MS spectra (5-8). In contrast, in shotgun lipidomics, total lipid extracts are infused directly into a mass spectrometer, and the molecular characterization of lipid species relies either on the accurately determined m/z of precursor ions (9) or on the detection of specific fragment ions or neutral losses in tandem mass spectrometric experiments (1, 9-12).Regardless of the analytical approach used, its success depends on the completeness of the extraction of lipids from corresponding cells, fluids, or tissues. Lipids of all major classes could be recovered via chloroform/methanol extraction, typically according to the Folch, Lees, and Sloane Stanley (13) or Bligh and Dyer (14) recipes (15), in which they are mostly enriched in the chloroform phase.Electrospray mass spectrometry, a major tool for analyzing complex lipidomes, is particularly sensitive towards the quality of lipid extracts. Coextracted components of biological matrix and salts (often, without further definition, termed background) affect both the sensitivity and specificity of lipid analysis. Often, abundant background ions obscure lipid precursors, and their MS/MS spectra are densely populated with "ghost" peaks and abundant chemical noise. Adducts with common background cations (sodium, potassium) and anions (chloride) increase the ambiguity of molecular species assignment and affect the accuracy of quantitative determination.Because of the higher density of chloroform compared with a water/methanol mixture, it forms the lower phase of the two-phase partitioning system. While collecting the chloroform fraction, a glass pipette or a needle of the pipetting robot reaches it through a voluminous layer...

Background/PurposeTo search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity.MethodsIn a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts.ResultsCompared with controls, the microbiome in patients with SLE showed decreased species richness diversity, with reductions in taxonomic complexity most pronounced in those with high SLE disease activity index (SLEDAI). Notably, patients with SLE had an overall 5-fold greater representation of Ruminococcus gnavus (RG) of the Lachnospiraceae family, and individual communities also displayed reciprocal contractions of a species with putative protective properties. Gut RG abundance correlated with serum antibodies to only 1/8 RG strains tested. Anti-RG antibodies correlated directly with SLEDAI score and antinative DNA levels, but inversely with C3 and C4. These antibodies were primarily against antigen(s) in an RG strain-restricted pool of cell wall lipoglycans. Novel structural features of these purified lipoglycans were characterised by mass spectrometry and NMR. Highest levels of serum anti-RG strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts.ConclusionThese findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.