Early experimental investigations of cellular heterogeneity used methods based on fluorescent proteins and antibodies, and these methods have been widely used for single-cell protein analysis over the last couple of decades. They have contributed to many foundational discussed in this review. However, these methods limit both the number of proteins that can be analyzed and the specificity of the analysis. Furthermore, these methods are limited in their ability to quantify similar proteoforms. These limitations may be overcome by extending mass-spectrometry analysis to the level of individual cells based on opportunities outlined in this perspective. These ideas have driven much exciting progress.         

Single-cell protein analysis Traditional methods identify and quantify a limited number of proteins based on antibodies barcoded with DNA sequences, fluorophores, or transition metals. Emerging single-cell mass-spectrometry (MS) methods will allow high-throughput analysis of proteins and their posttranslational modifications, interactions, and degradation.

Indeed, mass spectrometry is the most powerful method for protein analysis, but its application to single cells faces three major challenges: efficiently delivering proteins/peptides to mass spectrometry detectors, identifying their sequences, and scaling the analysis to many thousands of single cells. These challenges have motivated corresponding solutions, including isobaric carrier (SCoPE design) multiplexing and clean, automated, and miniaturized sample preparation. When synergistically applied, these solutions enable quantifying thousands of proteins across many single cells and have established a solid foundation for further advances. Building upon this foundation, the isobaric carrier concept will enable analyzing subcellular organelles and posttranslational modifications, while increases in multiplexing capabilities will increase the throughput and decrease cost.

These early methods for single-cell mass spectrometry analysis are posed for rapid growth and will help reveal mechanisms that underpin health and disease.