Quantification of Signaling Lipids by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI MS/MS).

Mathias Haag,Britta Brügger,Timo Sachsenheimer,Angelika Schmidt

doi:10.3390/metabo2010057

Abstract

Lipids, such as phosphoinositides (PIPs) and diacylglycerol (DAG), are important signaling intermediates involved in cellular processes such as T cell receptor (TCR)-mediated signal transduction. Here we report identification and quantification of PIP, PIP2 and DAG from crude lipid extracts. Capitalizing on the different extraction properties of PIPs and DAGs allowed us to efficiently recover both lipid classes from one sample. Rapid analysis of endogenous signaling molecules was performed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS), employing lipid class-specific neutral loss and multiple precursor ion scanning for their identification and quantification. Profiling of DAG, PIP and PIP2 molecular species in primary human T cells before and after TCR stimulation resulted in a two-fold increase in DAG levels with a shift towards 1-stearoyl-2-arachidonoyl-DAG in stimulated cells. PIP2 levels were slightly reduced, while PIP levels remained unchanged.

Highlights

Diacylglycerols (DAGs) and phosphoinositides (PIPs) are low abundant lipid classes in cellular membranes whose abundance is temporally and spatially tightly regulated
Protonation supports partitioning into the organic phase, which increases the recovery of low abundant PIPs
PLCγ during T cell activation [9], and PIP2 is re-synthesized by phosphorylation of PIP [55]

Summary

Introduction

Diacylglycerols (DAGs) and phosphoinositides (PIPs) are low abundant lipid classes in cellular membranes whose abundance is temporally and spatially tightly regulated. For the bio-synthesis of PIPs, the precursor phosphatidylinositol (PI) needs to be transported from the ER to different organelles, where PIPs are metabolically generated and interconverted by the enzymatic action of several kinases and phosphatases [1,2,3]. Dependent on the position and extent of phosphorylation, seven distinct phosphoinositide classes are known: PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3, with PI(4)P and PI(4,5)P2 being the most abundant forms. Some phosphoinositide classes can be regarded as organelle markers since they are predominantly synthesized and located at specific membranes, such as PI(3)P at early endosomes, PI(4)P at the Golgi complex and PI(4,5)P2 and PI(3,4,5)P3 at the plasma membrane [4]. The phosphorylated headgroup of PIPs binds to effector proteins and thereby triggers signaling and activation processes. These domains include pleckstrin homology (PH), phagocyte oxidase (PX), epsin N-terminal homology (ENTH), C2 and FYVE zinc finger domains [4,5]

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