17 research outputs found
The Ether Lipid Precursor Hexadecylglycerol Causes Major Changes in the Lipidome of HEp-2 Cells
<div><p>The ether-lipid precursor <i>sn-1</i>-O-hexadecylglycerol (HG) can be used to compensate for early metabolic defects in ether-lipid biosynthesis. To investigate a possible metabolic link between ether-linked phospholipids and the rest of the cellular lipidome, we incubated HEp-2 cells with HG. Mass spectrometry analysis revealed major changes in the lipidome of HG-treated cells compared to that of untreated cells or cells treated with palmitin, a control substance for HG containing an acyl group instead of the ether group. We present quantitative data for a total of 154 species from 17 lipid classes. These species are those constituting more than 2% of their lipid class for most lipid classes, but more than 1% for the ether lipids and glycosphingolipids. In addition to the expected ability of HG to increase the levels of ether-linked glycerophospholipids with 16 carbon atoms in the <i>sn-1</i> position, this precursor also decreased the amounts of glycosphingolipids and increased the amounts of ceramide, phosphatidylinositol and lysophosphatidylinositol. However, incubation with palmitin, the fatty acyl analogue of HG, also increased the amounts of ceramide and phosphatidylinositols. Thus, changes in these lipid classes were not ether lipid-dependent. No major effects were observed for the other lipid classes, and cellular functions such as growth and endocytosis were unaffected. The data presented clearly demonstrate the importance of performing detailed quantitative lipidomic studies to reveal how the metabolism of ether-linked glycerophospholipids is coupled to that of glycosphingolipids and ester-linked glycerophospholipids, especially phosphatidylinositols.</p></div
Long-Term Performance and Stability of Molecular Shotgun Lipidomic Analysis of Human Plasma Samples
The stability of the lipid concentration
levels in shotgun lipidomics
analysis was tracked over a period of 3.5 years. Concentration levels
in several lipid classes, such as phospholipids, were determined in
human plasma lipid extracts. Impact of the following factors on the
analysis was investigated: sample amount, internal standard amount,
and sample dilution factor. Moreover, the reproducibility of lipid
profiles obtained in both polarity modes was evaluated. Total number
of samples analyzed was approximately 6800 and 7300 samples in negative
and positive ion modes, respectively, out of which 610 and 639 instrument
control samples were used in stability calculations. The assessed
shotgun lipidomics approach showed to be remarkably robust and reproducible,
requiring no batch corrections. Coefficients of variation (CVs) of
lipid mean concentration measured with optimized analytical parameters
were typically less than 15%. The high reproducibility indicated that
no lipid degradation occurred during the monitored time period
Quantitative analysis of glycerophospholipids after HG or palmitin treatment.
<p>The major species of (<b>A</b>) PC O, (<b>B</b>) PC P, (<b>C</b>) PE O, (<b>D</b>) PE P, (<b>E</b>) PC and (<b>F</b>) PE, in HEp-2 cells treated with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) for 24 hours. The species shown here are species comprising more than 1% of the total mass of the ether lipids and more than 2% of PC and PE for at least one of the samples.</p
Overall changes in the lipidome of HEp-2 cells after treatment with HG or palmitin.
<p>HEp-2 cells were treated for 24 hours with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) before analyzing the lipidome by MS. (<b>A</b>) The total amount of the different lipid classes are shown as absolute values (note the logarithmic scale) and (<b>B</b>) the difference between treated cells and control cells are expressed as relative values.</p
Biosynthesis of ether and ester glycerophospholipids and the chemical structures of the precursors used.
<p>(<b>A</b>) Schematic overview of the biosynthesis of ether and ester glycerophospholipids. Note that 1-alkylglycerols such as HG (red box) may enter the pathway through phosphorylation to 1-alkylglycerol 3-phosphate by an alkylglycerol kinase. For abbreviations of the compounds and enzymes, see below. (<b>B</b>) The chemical structure of the compounds used in this study, HG and palmitin. DHAP; dihydroxyacetone phosphate, G3P; glycerol 3-phoshpate, DHAPAT; dihydroxyacetone phosphate acyltransferase, GPAT; glycerol phosphate acyltransferase, ADHAPS; alkyldihydroxyacetone phosphate synthase, LPA; lysophosphatidic acid, LPAAT; lysophosphatidic acid acyltransferase, PAP; phosphatidic acid phosphatase, EPT; ethanolamine phosphotransferase, CEPT; choline/ethanolamine phosphotransferase.</p
Quantitative analysis of ceramide and glycosphingolipids after HG or palmitin treatment.
<p>The major species of (<b>A</b>) Cer, (<b>B</b>) GlcCer, (<b>C</b>) LacCer, and (<b>D</b>) Gb3 in HEp-2 cells treated with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) for 24 hours. The species shown here are species comprising more than 1% of the total mass of any of the classes.</p
Quality control analysis.
<p>(<b>A</b>) Monitoring of lock mass offset and lock mass ion intensity as function of sample injection. Notice that the lock mass and internal standard TAG 17:1/17:1/17:1 is not detected in injection 07 and 08. Manual inspection of FT MS spectra revealed that the particular sample had not been spiked with internal standards. (<b>B</b>) Assessing the specificity of the PI species profile and intensity across all samples from wild-type mice and the negative control blank samples. Note that in the negative control blank sample (red) a low abundant background ion is detected and falsely identified as PI 40:3. Dubious lipid species can be removed using background subtraction and filtering during subsequent processing in Orange.</p
Lipidome visualization using different display formats.
<p>(<b>A</b>) mol% lipid category. Notice that the y-axis is logarithmic. Data is displayed as the average of the two technical replicates per sample. (<b>B</b>) mol% of db index of LPS species. Note that histogram include plot for both technical replicates. (<b>C</b>) mol% of PE species. Data is displayed as the average of the two technical replicates per sample. (<b>D</b>) mol% of all GPL species. Data is displayed as the average of the two technical replicates per sample. Notice that data for only one sample of cerebellum from knockout mice is available due to lack of spiked internal standards as outline in the section “Application of the ALEX software framework”. This neurolipidomics dataset is available as supporting information (Data S2).</p
Overview of the ALEX software framework and auxiliary workflow.
<p>The ALEX framework comprises six core modules (grey colored boxes). The function of each module is explained in the Results and Discussion section. The output of the ALEX framework includes a data file with identified lipid species, intensities and accessory lipid features across all processed samples and FT MS scan ranges. The ALEX output is organized in database table format that can be accessed and processed by the auxiliary workflow using Orange and Tableau software. The auxiliary workflow is designed to integrate sample information, compute lipid molar abundance, implement quality control procedures and visualize lipidome data.</p
The ALEX lipid calculator.
<p>(<b>A</b>) Representative positive ion mode FT MS spectrum of a 10:1-phase lipid extract of hippocampus from a PRG-1 knockout mouse. Note that the detection of selected lock mass ions tris(ditert-butylphenyl) phosphate (chemical background, [M+NH<sub>4</sub>]<sup>+</sup>, calculated <i>m/z</i> 680.48022, measured <i>m</i>/<i>z</i> 680.47945, <i>m</i>/<i>z</i> offset = -0.00077) and TAG 17:1/17:1/17:1 (internal standard (IS), [M+NH<sub>4</sub>]<sup>+</sup>, calculated <i>m/z</i> 860.77017, measured <i>m</i>/<i>z</i> 860.76889 and <i>m</i>/<i>z</i> offset = -0.00128). The FT MS calibration offset is estimated as the average of the <i>m/z</i> offset for both lock mass ions, i.e. the FT MS calibration offset = -0.0010. (<b>B</b>) Screenshot of the ALEX lipid calculator showing information for endogenous lipid species PC 32:0 while applying the FT MS calibration offset = -0.0010. Note that the measured <i>m/z</i> of PC 32:0 is 734.56872 and that the calculated <i>m/z</i> adjusted for the calibration offset is 734.56843 which yield a <i>m/z</i> difference of 0.00029 corresponding to a mass error of 0.4 ppm. Without applying lock mass adjustment the mass error would be 1 ppm.</p