Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics

Area of Science:

• Proteomics
• Cellular Biology
• Biochemistry

Background:

• Single-cell proteomics aims to enhance protein quantification consistency, sensitivity, and depth.
• Analyzing proteins and their modifications in individual cells presents significant technical challenges.

Purpose of the Study:

• To develop a method that simultaneously improves consistency, sensitivity, and depth in single-cell proteomics.
• To enable robust quantification of protein variation and functional covariation within and between cellular conditions.

Main Methods:

• Development of prioritized Single-Cell ProtEomics (pSCoPE) strategy.
• Analysis of thousands of prioritized peptides across single cells to maximize data completeness.
• Optimization of instrument time for analyzing identifiable peptides to increase proteome depth.

Main Results:

• pSCoPE achieved over twofold increase in sensitivity, data completeness, and proteome coverage.
• Quantified protein variation in untreated and lipopolysaccharide-treated primary macrophages.
• Identified covariation of proteins within functional sets (e.g., phagosome maturation) linked to phenotypic variability.

Conclusions:

• pSCoPE significantly advances single-cell proteomics capabilities.
• The method enables detailed analysis of protein covariation and functional relationships in cellular populations.
• pSCoPE is freely available and applicable for analyzing proteins of interest without compromising proteome coverage.