Fluorescence-Guided Matrix-assisted Laser Desorption/ Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry (MS) is capable of label-free, high-throughput characterization of endogenous molecules in individual cells. The recent advances in the development of MALDI mass spectrometers with laser-induced post-ionization (MALDI-2) provide greatly enhanced sensitivity of detection for a variety of lipids and other small molecules. However, MS imaging of large samples with MALDI-2 at cellular resolution is prohibitively slow for most applications. In this protocol, primary cells are isolated and dispersed onto conductive slides. Relative cell locations are determined by whole-slide fluorescence microscopy, followed by accurate coregistration of the microscopy coordinates to the stage coordinates of the MALDI-2 mass spectrometer. Targeted MS analysis of only cell locations provides high-throughput, single-cell measurements with high analyte coverage and reduced data size as compared to MS imaging of the entire sample. We describe the critical steps necessary for single-cell preparation, whole-slide fluorescence imaging, matrix application, and MALDI-2 mass spectrometry.