Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down level single cell. Experiments using this have achieved over 3,000 across 1,500 mammalian cells (500-1,000 per cell) in 10 days mass spectrometer instrument time. SCoPE2 leverages freeze-heat cycle cell lysis, obviating need clean-up consequently reducing sample losses, while expediting preparation simplifying its automation. Additionally, uses an isobaric carrier, which aids protein identification reduces losses. This video provides detailed guidance enable adoption automated single-cell only equipment reagents that are accessible. We demonstrate critical steps procedure preparing proteomic analysis, harvesting up injection liquid chromatography-tandem spectrometry (LC-MS/MS) analysis. viewers guided through principles experimental design with quality control both carrier preparations, representative results discussion limitations approach.