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• Schmidt, A
• Sachsenheimer, T
• Brügger, B
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• Schmidt, A
• Sachsenheimer, T
• Brügger, B
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• Schmidt, A
• Sachsenheimer, T
• Brügger, B

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Metabolites 2012, 2(1), 57-76; doi:10.3390/metabo2010057

Article

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

Mathias Haag ¹ , Angelika Schmidt ² , Timo Sachsenheimer ¹  and Britta Brügger ^1,*

¹ Heidelberg University Biochemistry Center, University of Heidelberg, Heidelberg, Germany ² Tumorimmunology Program, Division of Immunogenetics (D030), German Cancer Research Center (DKFZ), Heidelberg, Germany

* Author to whom correspondence should be addressed.

Received: 29 November 2011; in revised form: 4 January 2012 / Accepted: 6 January 2012 / Published: 16 January 2012

(This article belongs to the Special Issue Lipidomics)

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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.

Keywords: T cell signaling; signaling lipids; diacylglycerol; phosphoinositides; lipidomics; nano-electrospray ionization tandem mass spectrometry

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