Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Abstract

Changes in sphingolipids have been associated with profound effects in cell fate and development in both plants and animals. Sphingolipids as a group consist of a large number of different compound classes of which numerous individual species may vary in response to environmental stimuli to affect cellular responses. The ability to measure all sphingolipids simultaneously is, therefore, essential to an understanding of the biochemical regulation of sphingolipid metabolism and signaling molecules derived from it. In the model plant Arabidopsis thaliana, the major sphingolipid classes are glycosylinositolphosphoceramides, glucosylceramides, hydroxyceramides and ceramides. Other minor but potentially important sphingolipids are free long-chain bases and their phosphorylated derivates. By using a single solvent system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry detection we have been able to separate and measure 168 sphingolipids from a crude sample. This greatly speeds up and simplifies the analysis of plant sphingolipids and should pave the way for a better understanding of their role in plant performance.

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

“…In contrast, the smaller lipid pool of sugar-free ceramides contains non-hydroxylated forms to some extent. 69,70 This supports the possibility that activation of AtFAH by AtBI-1 contributes to enlargement of the glycosphingolipid pool. 56 The AtBI-1/AtFAH complex may have a role in the formation of membrane microdomains such as 'lipid raft'.…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…For example, over 90% of complex sphingolipids in Arabidopsis possess 2-HFAs (Markham and Jaworski, 2007), and hGlcCers and hGIPCs are abundant in rice, which unlike Arabidopsis also contains nGIPCs. In this study, double knockdown of Os-FAH1 and Os-FAH2 led to a decrease in 2-hydroxy (A) Immunoblot analysis of Rac1, PIP1s, and tubulin in wild-type and OsFAH1/2-KD1 suspension cells.…”

“…Analytical conditions described for Arabidopsis thaliana by Markham and Jaworski (2007) and Nagano et al (2014) were optimized for rice sphingolipid species. Chromatographic separation was performed using an XR-ODSII column (Shimadzu; 2.2 mm, 2.0 mm ID, 75 mm) held at 40°C with binary elution gradients consisting of THF/methanol/10 mM ammonium formate (3:2:5) containing 0.1% (v/v) formic acid as solvent A and THF/methanol/10 mM ammonium formate (7:2:1) containing 0.1% (v/v) formic acid as solvent B at a flow rate of 200 mL/min.…”

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activities

“…Sphingolipids were extracted from 2 to 15 mg of lyophilized plant material and analyzed by liquid chromatography-tandem mass spectrometry as described previously (Markham and Jaworski, 2007;Kimberlin et al, 2013).…”