# SCoPE2 protocol

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying over 1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Isobaric carrier based multiplexed single-cell proteomics is a scalable, reliable, and cost-effective method that can be fully automated and implemented on widely available equipment. It uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. Here we describe an automated SCoPE2 workflow that allows analyzing about 200 single cells per 24 hours using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

## Data

• RAW Files

• Peptides x Samples

• Peptides x single cells at 1% FDR. The first 2 columns list the corresponding protein identifiers and peptide sequences and each subsequent column corresponds to a single cell. Peptide identification is based on spectra analyzed by MaxQuant and is enhanced by using DART-ID to incorporate retention time information. See Specht et al., 2019 for details.

• Peptides x Samples
• Proteins x single cells at 1% FDR, imputed and batch corrected.

• Cells.txt
• Annotation x single cells. Each column corresponds to a single cell and the rows include relevant metadata, such as, cell type if known, measurements from the isolation of the cell, and derivative quantities, i.e., rRI, CVs, reliability.

• Source data for figures

## Workshops

The presentations below describe sample preparation and data analysis for the latest version of Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), SCoPE2. They were presented at the workshop of the second Single Cell Proteomics Conference.