Low-Input Proteomic Analysis of In Situ Fixed Adult Murine Muscle Stem Cells

Preserving the quiescent state of adult stem cells is challenging because they rapidly activate in response to external stimuli. Conventional isolation methods for muscle stem cells (MuSCs), such as enzymatic digestion followed by fluorescence-activated cell sorting (FACS), are time-consuming, resulting in early activation and loss of quiescent features. Here, we introduce an in situ fixation protocol for the dissociation and proteomic characterization of quiescent MuSCs using trapped ion-mobility time-of-flight (timsTOF) mass spectrometry. This protocol encompasses mouse perfusion, FACS preparation and sorting, protein extraction, decrosslinking, digestion, and subsequent liquid chromatography-mass spectrometry (LC-MS) analysis. Our approach effectively preserves the quiescent features of MuSCs, enabling precise proteomic analysis with limited sample amounts and providing novel insights into stem cell biology.