Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of N-Acyl Moiety of Sphingomyelin

  • Lipids. 2017 Sep;52(9):789-799. doi: 10.1007/s11745-017-4279-5.
Kotaro Hama  1 ,  Yuko Fujiwara  2 ,  Hidetsugu Tabata  2 ,  Hideyo Takahashi  2 ,  Kazuaki Yokoyama  2
Affiliations
  • 1. Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan. [email protected].
  • 2. Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan.
PMID: 28770378 DOI: 10.1007/s11745-017-4279-5
Abstract

Sphingomyelin (ceramide-phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N-acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N-acyl fatty acids as [RCO2]- ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof-of-principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC-ESI-MS/MS/MS analysis.

Keywords
Liquid chromatography-electrospray ionization-tandem mass spectrometry; MS/MS/MS analysis; N-Acyl fatty acid; Sphingomyelin.
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