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.
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.