Retention Time Prediction Improves Identification in Nontargeted Lipidomics Approaches

Identification of lipids in nontargeted lipidomics based on liquid-chromatography coupled to mass spectrometry (LC-MS) is still a major issue. While both accurate mass and fragment spectra contain valuable information, retention time (<i>t</i>_R) information can be used to augment this data. We present a retention time model based on machine learning approaches which enables an improved assignment of lipid structures and automated annotation of lipidomics data. In contrast to common approaches we used a complex mixture of 201 lipids originating from fat tissue instead of a standard mixture to train a support vector regression (SVR) model including molecular structural features. The cross-validated model achieves a correlation coefficient between predicted and experimental test sample retention times of <i>r</i> = 0.989. Combining our retention time model with identification via accurate mass search (AMS) of lipids against the comprehensive LIPID MAPS database, retention time filtering can significantly reduce the rate of false positives in complex data sets like adipose tissue extracts. In our case, filtering with retention time information removed more than half of the potential identifications, while retaining 95% of the correct identifications. Combination of high-precision retention time prediction and accurate mass can thus significantly narrow down the number of hypotheses to be assessed for lipid identification in complex lipid pattern like tissue profiles

Accurate Sphingolipid Quantification Reducing Fragmentation Bias by Nonlinear Models

Quantitative sphingolipid analysis is crucial for understanding the roles of these bioactive molecules in various physiological and pathological contexts. Molecular sphingolipid species are typically quantified using sphingoid base-derived fragments relative to a class-specific internal standard. However, the commonly employed “one standard per class” strategy fails to account for fragmentation differences presented by the structural diversity of sphingolipids. To address this limitation, we developed a novel approach for quantitative sphingolipid analysis. This approach utilizes fragmentation models to correct for structural differences and thus overcomes the limitations associated with using a limited number of standards for quantification. Importantly, our method is independent of the internal standard, instrumental setup, and collision energy. Furthermore, we integrated this method into a user-friendly KNIME workflow. The validation results illustrate the effectiveness of our approach in accurately quantifying ceramide subclasses from various biological matrices. This breakthrough opens up new avenues for exploring sphingolipid metabolism and gaining insights into its implications

UNC5B expression in murine tissue.

<p><b>A)</b> Relative protein expression in murine brain (Br), intestine (In), liver (Li), heart (He) and kidney (Ki) by Western Blot analysis (n = 3; pooled samples). <b>B)</b> Gating of leukocytes subpopulations such as lymphocytes, monocytes and neutrophil granulocytes. <b>C)</b> Percentage of positive gated neutrophil granulocytes, leukocytes, monocytes and T- and B-cells (n = 5). Representative histograms of UNC5B expression (red) of CD45+ leukocytes and CD15+ neutrophils are shown <b>D)</b> Immunofluorescence UNC5B (green = Alexa 488) co-staining on neutrophil leukocytes (CD45 marked) and granulocytes (CD15 marked). Merged images show the localization of UNC5B on the neutrophils and leukocytes (n = 3 per group).</p

SiRNA in-vivo knockdown of UNC5B attenuates myocardial IR injury in WT mice.

<p><b>A)</b> Infarct size of mice with UNC5B specific siRNA treatment compared to mice received non-targeting siRNA after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice (n = 6 per group; *<i>P</i><0.05; ***<i>P</i><0.001 as indicated). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the siRNA treated mice.</p

Myocardial infarction is diminished in <i>UNC5B^+/−</i> mice.

<p><b>A)</b> Comparison of UNC5B protein expression in <i>UNC5B^+/−</i> and WT animals in different organs (n = 5 per group). <b>B)</b> Comparison of UNC5B transcriptional mRNA levels of organs normalized to brain tissue of WT animals (n = 5 per group). <b>C)</b> Infarct size in <i>UNC5B^+/−</i> mice compared with WT mice after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. <b>D)</b> and <b>E)</b> Correlating serum troponin I and IL-6 levels of <i>UNC5B^+/−</i> and WT mice (n = 6 per group; *<i>P</i><0.05; **<i>P</i><0.01 as indicated) <b>F)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infarcted tissue).</p

Functional inhibition of UNC5B reduces damage in myocardial IR.

<p><b>A</b>) Infarct size after IR in WT mice receiving antibody against UNC5B (2.5 µg/mouse) iv. 30 min before onset of surgery. Controls were treated with corresponding IgG antibody. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice. (n = 6 per group; *<i>P</i><0.05; ***<i>P</i><0.001 as indicated). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the antibody treated mice.</p

Functional inhibition of UNC5B after depletion of neutrophil granulocytes does not results not in additional cardioprotection.

<p><b>A)</b> Infarct size following IR in WT and UNC5B^+/− mice after neutrophil granulocyte depletion and subsequent anti- UNC5B antibody adminsitration. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice. (n = 4 per group). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted necrotic tissue.</p

Insulin induces binding of 14-3-3 to endogenous IRS-2 and phosphorylates Ser-573 on IRS-2.

<p><i>A.</i> Fao cells were starved for serum overnight and incubated for 30 min with either 10 nM insulin or 50 ng/ml IGF-1. 250 µg protein was immunoprecipitated with IRS-2 antibody and separated on a 5–15% gradient gel. Overlay assay followed stripping and reprobing with IRS-2 antibody as loading control. Successful stimulation is shown as phosphorylation of p-Thr-308 and corresponding Akt/PKB reblot. <i>B.</i> Fao cells were starved for serum overnight and stimulated with 10 nM insulin for the indicated time points. 100 µg of protein was separated on a 7.5% gel and membranes were probed with specific antibodies against p-Ser-573 of IRS-2 and p-Thr-308 of Akt/PKB. For loading control membranes were stripped and reprobed with protein antibody.</p

Transendothelial neutrophil migration is inhibited by anti- UNC5B antibody.

<p><b>A</b>) PMN transmigration studies were performed in transwells using fMLP as chemoattractant in the lower compartment. PMNs were pre-incubated with either anti-UNC5B antibody or IgG control prior to transmigration. Number of transmigrated PMNs was assessed by MPO measurement. <b>B</b>) Passive flux of FITC Dextran across HMEC endothelium monolayers. Flux was detected by measurement of passed FITC Dextran in the basal compartment (n = 6 per condition; *<i>P</i><0.05 as indicated).</p

Co-immunoprecipitation and overlay assays indicate interaction of 14-3-3 and IRS-2 upon IGF-1/insulin stimulation.

<p><i>A.</i> HEK293 cells were transiently transfected with GFP or GFP-IRS2 and after serum starvation cells were incubated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. 400 µg total protein was used for immunoprecipitation with 14-3-3 antibody (C-17) and samples were separated on 5–15% gradient gel. Upper membrane was incubated with IRS-2 antibody, lower membrane with 14-3-3 antibody (K-19). <i>B.</i> HEK293 cells were transfected with GFP-IRS2 and stimulation was carried out after starvation for serum overnight with 50 ng/ml IGF-1 for 30 min or subsequently after preincubation with 1 µM PI-103 for 30 min. 250 µg of total protein was pulled down using GFP-Trap®. SDS-PAGE followed transfer onto nitrocellulose membranes. Overlay assay followed stripping of the membrane and reprobing with GFP antibody as loading control. <i>C.</i> Extent of interaction was quantified by scanning densitometry of blots and normalization for GFP-IRS2 serum starved condition (mean ± SEM; n = 4; *p<0.05 serum starved vs. IGF-1 or IGF-1 vs. PI-103/IGF-1). <i>D</i>. Male C57Bl/6 mice were fasted overnight and injected intravenously with 2 IU (international units) insulin. After 10 min liver was taken and 500 µg of total protein was immunoprecipitated with IRS-2 antibody. After performing overlay assay membrane was stripped and reprobed with IRS-2 antibody as loading control. Two mice of each group are shown. <i>E</i>. Densitometric analyses of 14-3-3 interaction with IRS-2. Overlay signal was normalized for total IRS-2 protein content (mean ± SEM; n = 4; *p<0.05 fasted vs. insulin). <i>F</i>. Male C57Bl/6 mice were fasted overnight, refed for 4 hours or injected intraperitoneally with insulin for 30 min. Procedure as in <i>G</i>. Four mice of each group are shown. <i>G</i>. 14-3-3 interaction with IRS-2 was quantified by scanning densitometry of immunoblots and normalization for IRS-2 protein (mean ± SEM; n = 4; *p<0.05 fasted vs. refed and insulin stimulation).</p