Standardized Workflow for Mass-Spectrometry-Based Single-Cell Proteomics Data Processing and Analysis Using the scp Package.

Mass-spectrometry (MS)-based single-cell proteomics (SCP) explores cellular heterogeneity by focusing on the functional effectors of the cells-proteins. However, extracting meaningful biological information from MS data is far from trivial, especially with single cells. Currently, data analysis workflows are substantially different from one research team to another. Moreover, it is difficult to evaluate pipelines as ground truths are missing. Our team has developed the R/Bioconductor package called scp to provide a standardized framework for SCP data analysis. It relies on the widely used QFeatures and SingleCellExperiment data structures. In addition, we used a design containing cell lines mixed in known proportions to generate controlled variability for data analysis benchmarking. In this chapter, we provide a flexible data analysis protocol for SCP data using the scp package together with comprehensive explanations at each step of the processing. Our main steps are quality control on the feature and cell level, aggregation of the raw data into peptides and proteins, normalization, and batch correction. We validate our workflow using our ground truth data set. We illustrate how to use this modular, standardized framework and highlight some crucial steps.