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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bcraiv2e

Authors: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Chris Anderton, Kumar Sharma

Group: KPMP, Metabolomics Protocols & Workflows

Summary: Mass spectrometry imaging (MSI) is a cutting-edge molecular technology that enables simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs. For MSI, cryosections are prepared from flash-frozen tissue and mounted on a contuctive glass slide. We use matrix-assisted laser desorption ionization (MALDI)-MSI, where tissue sections are coated with a MALDI matrix in order to facilitate laser ionization and detection of metabolites with mass spectrometry. We do it using an automated robotic sprayer (TM-Sprayer) with 2,5- dihydroxybenzoic acid (DHB) MALDI matrix for positive ion mode analysis, N-(1-naphthyl) ethylenediamine hydrochloride (NEDC) for negative ion mode analyses, or 1,5-Diaminonaphthalene (DAN) for dual-polarity analysis, prior to being loaded into the MALDI-Q Exactive HF-X Orbitrap-MSI or MALDI-FTICR-MSI for untargeted metabolomics analysis. We have optimized a method for matrix application to maximize analyte extraction from tissue and increase MALDI sensitivity and to create the most homogenous matrix films possible for best lateral resolution of lipids. A key capacity that will be critical for scale-up in KPMP is the use of a TM-sprayer robotic sprayer for MALDI matrix application. This will make inter-lab studies viable, by increasing the reproducibility of sample preparation and is currently in use at UTHSA and PNNL. Our lipid extraction protocol for LC-MS/MS is a robust and universal protocol for single-sample integrative proteomics, metabolomics, and lipidomics analyses (see citation).

Affiliations: University of Texas Health San Antonio, Pacific Northwest National Laboratory, University of Texas Health San Antonio, Pacific Northwest National Laboratory, European Molecular Biology Laboratory, Pacific Northwest National Laboratory, University of Texas Health San Antonio

Version: 1

Publication Date: 2020

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