In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry
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The neuromuscular junction (NMJ), where a motor neuron intercepts and activates a muscle fiber, is a highly versatile and complex subcellular region. Genomic and proteomic approaches using the large (>1 kg) electric organ of Torpedo californica have helped advancing our understanding of this minute (30–50 μm) electric synapse. However, the majority of these studies have focused on mRNA and proteins, therefore neglecting small signaling molecules involved in muscle-nerve ‘dialogue’. We developed a novel technique, mid-infrared laser ablation electrospray ionization (LAESI) mass spectrometry (MS), with the potential of detecting a diversity of small signaling molecules in vitro. LAESI uses the native water in the tissue as the matrix to couple the laser pulse energy into the target for the ablation process and enables its direct analysis essentially without sample preparation. Here, we report the detection of metabolites from the untreated frozen tissue of the Torpedo electric organ with LAESI MS at atmospheric pressure. A total of 24 metabolites were identified by accurate mass measurements, natural isotope patterns, and tandem mass spectrometry. Most of the identified metabolites were related to the cholinergic function of the electric synapse (acetylcholine and choline), fatty acid metabolism and acetyl transfer (carnitine and acetylcarnitine), the mitigation of osmotic stress (betaine and trimethylamine N-oxide), and energy production (creatine and creatinine). The biosynthetic precursors of these metabolites and their expected degradation products were also detected indicating that LAESI MS is well suited for tissue metabolomics with the ultimate goal of imaging and in vivo studies.
KeywordsTorpedo californica Electric organ Neuromuscular junction Metabolites Osmolytes Metabolomics Quantitation Laser ablation Electrospray ionization LAESI Collision activated dissociation
The authors are grateful for the support of this work by the W. M. Keck Foundation (041904), the National Science Foundation under grant 0719232, and the Research Enhancement Fund of the George Washington University. The opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors thank Peter Nemes (George Washington University) for his help in setting up the LAESI experiments. One of the authors (P. S.) thanks the Director of the Indian Institute of Chemical Technology, Hyderabad, and the Council of Scientific and Industrial Research, India for granting leave.
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