Ganglioside lipidomics of CNS myelination using direct infusion shotgun mass spectrometry.

Gangliosides are present and concentrated in axons and implicated in axon-myelin interactions, but how ganglioside composition changes during myelin formation is not known. Here, we present a direct infusion (shotgun) lipidomics method to analyze gangliosides in small amounts of tissue reproducibly and with high sensitivity. We resolve the mouse ganglioside lipidome during development and adulthood and determine the ganglioside content of mice lacking the St3gal5 and B4galnt1 genes that synthesize most ganglioside species. Our results reveal substantial changes in the ganglioside lipidome during the formation of myelinated nerve fibers. In sum, we provide insights into the CNS ganglioside lipidome with a quantitative and sensitive mass spectrometry method. Since this method is compatible with global lipidomic profiling, it will provide insights into ganglioside function in physiology and pathology.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Correction of Isobaric Overlap Resulting from Sodiated Ions in Lipidomics.

Lipidomic analyses aim for absolute quantification of lipid species profiles in biological samples. In past years, mass spectrometry (MS) methods based on high resolution accurate masses (HRAM) have increasingly been applied to identify and quantify lipid species on the MS level. This strategy requires consideration of isobaric overlaps which may also result from various adduct ions. Generally applied solvent additives favor the formation of protonated and ammoniated ions in positive ion mode, yet sodiated ions are also frequently observed. These sodiated ions interfere with protonated ions of the species of the same lipid class with two additional CH2 and three double bonds (Δm/z = 0.0025) and the first isotopic peak overlaps with ammoniated ions of a species with one additional CH2 and four double bonds (Δm/z = 0.0057). In this work, we present an algorithm based on the sodiated to protonated/ammoniated adduct ion ratios of applied internal standards to correct for these interferences. We could demonstrate that these ratios differ significantly between lipid classes but are affected by neither chain length nor number of double bonds within a lipid class. Finally, the algorithm is demonstrated for correcting human serum samples analyzed by Fourier-transform mass spectrometry (FTMS). Here, the application of sodium correction significantly reduced overestimations and misidentifications.

Gravimetric determination of phospholipid concentration.

Accurate determination of lipid concentrations is an obligatory routine in a research laboratory engaged in studies using this class of biomaterials. For phospholipids, this is frequently accomplished using the phosphate assay (Bartlett, G.R. Phosphorus Assay in Column Chromatography. J. Biol. Chem. 234, 466-468, 1959). Given the purity of the currently commercially available synthetic and isolated natural lipids, we have observed that determination of the dry weight of lipid stock solutions provides the fastest, most accurate, and generic method to assay their concentrations. The protocol described here takes advantage of the high resolution and accuracy obtained by modern weighing technology. We assayed by this technique the concentrations of a number of phosphatidylcholine samples, with different degrees of acyl chain saturation and length, and in different organic solvents. The results were compared with those from Bartlett assay, (31)P NMR, and Langmuir compression isotherms. The data obtained show that the gravimetric assay yields lipid concentrations with a resolution similar or better than obtained by the other techniques.

Extensive long-distance pollen dispersal in a fragmented landscape maintains genetic diversity in white spruce.

Conifers are among the most genetically diverse plants but show the lowest levels of genetic differentiation, even among geographically distant populations. High gene flow among populations may be one of the most important factors in maintaining these genetic patterns. Here, we provide empirical evidence for extensive pollen-mediated gene dispersal between natural stands of a widespread northern temperate/boreal conifer, Picea glauca. We used 6 polymorphic allozyme loci to quantify the proportion of seeds sired by pollen originating from different sources in a landscape fragmented by agriculture in North Central Ontario, Canada. In 7 stands, a small proportion of seeds were sired by self-pollen or neighboring trees but 87.1% (+/-1.7% standard error [SE]) of seeds were sired by pollen from at least 250 to 3000 m away. In 4 single isolated trees, self-fertilization rates were low and more than 96% (+/-1.3% SE) of seeds were sired by immigrant pollen. The average minimum pollen dispersal distance in outcrossed matings was 619 m. These results provide strong evidence that extensive long-distance pollen dispersal plays a primary role in maintaining low genetic differentiation among natural populations of P. glauca and helps maintain genetic diversity and minimize inbreeding in small stands in a fragmented landscape.