Single-cell protein analysis by mass spectrometry

Abstract

Human physiology and pathology arise from the coordinated interactions of diverse single cells. However, analyzing single cells has been limited by the low sensitivity and throughput of analytical methods. DNA sequencing has recently made such analysis feasible for nucleic acids but single-cell protein analysis remains limited. 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 SCoPE design multiplexing and clean, automated, and miniaturized sample preparation. Synergistically applied, these solutions enable quantifying thousands of proteins across many single cells and establish a solid foundation for further advances. Building upon this foundation, the SCoPE concept will enable analyzing subcellular organelles and posttranslational modifications, while increases in multiplexing capabilities will increase the throughput and decrease cost.

Keywords: Isobaric carrier; Mass-spectrometry; Sample preparation; Single-cell analysis; Single-cell proteomics; Systems biology.

Figure 1|. Major challenges to single-cell protein analysis by mass-spectrometry and their solutions

Single-cell proteomics by mass-spectrometry faces three main challenges: (i) Delivering enough copy number of ions to MS detectors to afford accurate quantification; (ii) Reliable amino acid sequence determination of quantified ions; (iii) Scaling the analysis to many thousands of single cells at affordable cost. Each of these challenges is addressed by one or more solutions. These solutions are highly synergistic and thus depicted as interlocking puzzle pieces with a few prominent examples from each category.

Figure 2|. Conceptual work flow of automated sample preparation, the isobaric carrier design, enhanced peptide sequence identification, and LC-MS/MS optimization as implemented by SCoPE2

SCoPE2provides solutions for all challenges from Figure 1: (i) Protein delivery for MS analysis is facilitated by the clean, automated and miniaturized lysis by mPOP [35], and by the isobaric carrier design, which combines isobarically-labeled peptides from single-cell and from carrier samples [32, 13], 33. (ii) Peptide sequence identification is enhanced by the carrier peptides contributing fragment ions to the MS2 spectra and by DART-ID [36]. (iii) Analysing many cells is facilitated by fully automated sample preparation and analysis, and by the isobaric carrier design multiplexing with TMT pro [35, 4], 5, 13.