An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes

This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversedphase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of similar to 584,000 unique peptide sequences and similar to 14,200 protein isoforms (similar to 12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including similar to 7,000 N-acetylation sites and similar to 10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.