Recently single-cell mass-spectrometry analysis has allowed quantifying thousands of proteins in single mammalian cells. Yet, these technologies have been adopted in relatively few mass-spectrometry laboratories. Increasing their adoption can help reveal biochemical mechanisms that underpin health and disease, and it requires robust methods that can be widely deployed in any mass spectrometry laboratory.
This aim for a “model T” single-cell proteomics has been the guiding philosophy in the development of Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) and its version 2 (SCoPE2). We aimed to make every step easy to reproduce, from sample preparation and experimental parameters optimization to an open source data analysis pipeline. The emphasis has been on accuracy and accessibility, which has facilitated replication and adoption of SCoPE2. Yet, we still found that some groups adopting these single-cell technologies fail to make quantitatively accurate protein measurements, because they skip important quality control steps of sample preparation (such as negative controls and labeling efficiency), and mass spectrometry analysis, such as apex sampling and purity of MS2 spectra.
These observations motivated us to write a detailed protocol for multiplexed single-cell proteomics. The protocol emphasizes quality controls that are required for accurately quantifying protein abundance in single cells and scaling up the analysis to thousands of single cells. The protocol and its associated video and web resources should make single-cell proteomics accessible to the wider research community.