Marín-Vicente, ConsueloCalvo, EnriqueRodríguez, José ManuelVilla Del Campo, CristinaSierra, RocíoVégvári, ÁkosZubarev, Roman ATorres, MiguelVázquez, Jesús2025-12-162025-12-162024Methods Mol Biol. 2024:2817:33-43.https://hdl.handle.net/20.500.12105/27057Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.engAMMass spectrometryMultiplex sample preparation strategyOrbitrap Eclipse mass spectrometerSingle-cell proteomicsTandem mass tagTribrid mass spectrometer38907145Methods in molecular biology (Clifton, N.J.)open access