High-throughput Fluorescence-guided Sequential Single-Cell MALDI-ICC Mass Spectrometry

We developed a sequential single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) workflow that enables endogenous lipid profiling in the first step, followed by cell-type classification of the same cells via immunocytochemistry in the second step. This stepwise approach integrates high-throughput single-cell analysis enabled by microMS with multiplex immunolabeling using photocleavable mass tags (PCMTs), which are antibodies conjugated to peptide mass reporters that are photoreleased and then detected by MALDI-MS. This platform combines the strengths of untargeted chemical profiling with targeted marker-based cell identification, allowing characterization of the cells’ endogenous metabolic activity, followed by cell classification using well-established immunomarkers. Here, we provide the raw data, mzML-converted files, and LC-MS/MS data from rodent hippocampal cells as described in the manuscript.