Sphingolipid Analysis by High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS)

Abstract

S phingolipid (SPL) metabolism ( Fig. 1) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosine1-phosphate (S1P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary.Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information.

Bone-specific insulin resistance disrupts whole-body glucose homeostasis via decreased osteocalcin activation

“…Samples were centrifuged at 300 g for 5 minutes to remove debris, and supernatants were transferred to fresh microtubes. Aliquots of homogenate were removed for estimation of protein concentration by the Bradford assay, and separate aliquots (2-300 μl containing a range of 0.45 to 0.8 mg protein) were analyzed for DAG levels by tandem LC/MS mass spectrometry at the Medical University of South Carolina Lipidomics Core, as described previously (53). Lipid levels measured (pmol) were normalized to cellular protein, and total lipid levels reflect the sum of all measured species.…”

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line

“…The pellets were lysed in RIPA buffer and an aliquot used for protein estimation by BCA method (38) and the remainder kept frozen at 80°C until used for sphingolipidomic analysis. A HPLC-MS/MS analysis of endogenous Cer, dihydroceramide (dhCer), and SM components was performed on a ThermoFisher TSQ Quantum or SCIEX Q-Trap triple-stage quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode, as previously described (39,40). Briefly, test samples were fortified with the internal standards (ISs) , and extracted into a one-phase solvent system with ethyl acetate/ isopropanol/water (60/30/10%; v/v).…”

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes

“…Medium from hepatocytes treated with palmitate or vehicle was analyzed for S1P content, as previously described ( 25 ).…”