Journal: Metabolites

Article Title: Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics.

doi: 10.3390/metabo10090375

Figure Lengend Snippet: Figure 2. Overview of the GIPC fragmentation for the example of GIPC A-OH-t18:1/h24:0 in salad: (A) The fragment assignment of GIPC A-OH-t18:1/h24:0 (adapted from [23]). The W fragment is shown in a light blue color. Please note that a full structural characterization is not possible by RP-HRMS/MS, (B) The product ion spectrum in negative ion mode at m/z 1260.7237, showing characteristic fragments m/z 241 and 259, 355, 373 and 417. The sugar head group was confirmed by the [C3PO3]−fragment (m/z 597, R1 = OH). [Z0PO3]−and [Y1-H]−fragments prove the ceramide moiety. (C) The positive ion mode ddMS2 spectrum of the [M + H]+ precursor, exhibiting the [W]+, [W-H2O]+ and [W-2H2O]+ fragments at m/z 298, 280 and 262, which are characteristic for the t18:1 LCB.

Article Snippet: C16 Lactosyl(ß) Ceramide (d18:1/16:0) (D-lactosyl-ß-1,1’ N-palmitoyl-D-erythro-sphingosine) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Albama, USA), was used as internal standard (IS) and dissolved in an appropriate amount of IPA to achieve a concentration of 100 μM.

Techniques:

Journal:

Article Title: IRF8 Regulates Acid Ceramidase Expression to Mediate Apoptosis and Suppresses Myelogeneous Leukemia

doi: 10.1158/0008-5472.CAN-10-2493

Figure Lengend Snippet: Ceramide contents were determined by LC/MS. Shown are representative results of two independent measurements.

Article Snippet: Measurement of apoptotic cell death Cells were treated with either IFN-γ (R&D Systems) overnight, acid ceramidase inhibitor LCL85 overnight, or C16 ceramide (Santa Cruz) for 1 h, followed by incubation with recombinant FasL (100 ng/ml, PeproTech; Rocky Hill, NJ) for approximately 24 h. Cells were then collected and incubated with propidium iodide (PI) solution (R&D Systems) and analyzed by flow cytometry.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal:

Article Title: IRF8 Regulates Acid Ceramidase Expression to Mediate Apoptosis and Suppresses Myelogeneous Leukemia

doi: 10.1158/0008-5472.CAN-10-2493

Figure Lengend Snippet: A. Restoration of IRF8 expression sensitizes CML cells to Fas-mediated apoptosis. 32D.Vector and 32D.IRF8 cells were cultured in the absence and presence of recombinant FasL for approximately 24 h and analyzed for apoptosis. Column, mean; Bar, SD. **p<0.01. Shown are representative results of one of three independent experiments. B. Inhibition of A-CDase activity increased CML cell sensitivity to Fas-mediated apoptosis. 32D-BA cells were either untreated (control) or treated with A-CDase inhibitor LCL85 overnight, followed by incubation with FasL for approximately 24 h and then analysis for apoptosis. Shown are representative images of three separate experiments. Column, mean; Bar, SD. ** p<0.01. C. Exogenous C16 ceramide increases CML cell sensitivity to Fas-mediated apoptosis. 32D-BA cells were cultured in the presence of C16 ceramide for 1 h, followed by incubation with FasL for approximately 16 h. Cell death was measured as in A. D. Silencing A-CDase or Overexpressing A-CDase alters CML cells sensitivity to Fas-mediated apoptosis. Top panel: 32D-BA cells were transduced with lentivirus containing either scramble shRNA or A-CDase-specific shRNA. The cells were then analyzed for A-CDase silencing efficiency by RT-PCR (left panel) and sensitivity to Fas-mediated apoptosis as described above in A. Shown are representative results of two independent experiments. Bottom panel: 32D-BA.IRF8 cells were transiently transfected with pEGFP vector or pEGFP-A-CDase and analyzed for A-CDase expression by RT-PCR (left panel) and sensitivity to Fas-mediated apoptosis (right panel) as described above in A. Shown are representative results of three independent experiments.

Article Snippet: Measurement of apoptotic cell death Cells were treated with either IFN-γ (R&D Systems) overnight, acid ceramidase inhibitor LCL85 overnight, or C16 ceramide (Santa Cruz) for 1 h, followed by incubation with recombinant FasL (100 ng/ml, PeproTech; Rocky Hill, NJ) for approximately 24 h. Cells were then collected and incubated with propidium iodide (PI) solution (R&D Systems) and analyzed by flow cytometry.

Techniques: Expressing, Plasmid Preparation, Cell Culture, Recombinant, Inhibition, Activity Assay, Control, Incubation, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Biomolecules

Article Title: Sperm Lipid Composition in Early Diverged Fish Species: Internal vs. External Mode of Fertilization.

doi: 10.3390/biom10020172

Figure Lengend Snippet: Figure 4. High performance thin-layer chromatography (HPTLC)-electrospray ionization ion trap (ESI-IT) mass spectrometry of the unknown spots of the lipid fraction of sterlet sperm. The chromatographic separation of the lipid extract and the corresponding mass spectra of two unknown spots in the positive and in the negative ion mode, respectively, are shown in (a). To further elucidate the structure of m/z 722, this ion was subsequently fragmented by collision-induced decay (b). Due to characteristic fragments with the loss of a hexose moiety (∆= 162 and 180) m/z 722 was assigned to a neutral glycosphingolipid Hex-Cer(d18:1/16:0). Hex: hexose, Cer: ceramide. * not assigned.

Article Snippet: C16 galactosyl(α) ceramide (d18:1/16:0) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was used as reference.

Techniques: High Performance Thin Layer Chromatography, Mass Spectrometry

Journal: Biomolecules

Article Title: Sperm Lipid Composition in Early Diverged Fish Species: Internal vs. External Mode of Fertilization.

doi: 10.3390/biom10020172

Figure Lengend Snippet: Figure 5. HPTLC-ESI-IT MS of unknown spots of the lipid fraction of stingray sperm. The chromatographic separation of the lipid extract and the corresponding positive and negative ion mass spectra of two unidentified spots are shown in (a). To further elucidate the structure of m/z 824 in the positive and m/z 778 in the negative ion mode, these ions were fragmented by collision-induced decay (b). Due to the detectability in the positive and the negative ion mode and the characteristic fragments with the loss of a sulfate group (∆= 80) and the detection of a sulfated sugar moiety in the negative ion mode (m/z 259 and 241), both signals were assigned to an acidic glycosphingolipid sulpho-Gal-Cer(d18:1/16:0). The signals at m/z 838/792, 840/794, and 854/808 could be putatively assigned to sulpho-Gal-Cer(d18:1/17:0), sulpho-Gal-Cer(d18:0/17:0), and sulpho-Gal-Cer(d18:0/18:0), respectively. Gal—galactose, Cer—ceramide. The assignment of the hexose unit to galactose was made due to the characteristic biosynthesis pathway of sulfo glycolipids [51]. * not assigned.

Article Snippet: C16 galactosyl(α) ceramide (d18:1/16:0) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was used as reference.

Techniques: High Performance Thin Layer Chromatography