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

Abstract

SummaryThis 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 reversed-phase 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 ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼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.

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

“…For example, two dimensional chromatography fractionation (8,9) and increased reverse phase column length (10,11) were reported to enhance peak capacity for proteome analyses. Improvements in quadrupole performance through higher resolution and scanning rate have enabled more efficient isolation and transmission of target ions with narrow isolation windows in turn reducing chimeric tandem mass spectra (12).…”

Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics

“…26 Using a modified offline high-pH fractionation technique, Bekker-Jensen et al demonstrated that proteomic analysis can reach a similar depth as the next-generation RNA-seq technology. 25 These approaches reduced the complexity of peptide mixtures and enabled deep analysis of cellular proteomes. Substantial increments of instrument time and sample consumption are needed to compensate for the increased number of protein IDs, which not only reduce the throughput of the experiment, but also impede the automation of the entire procedure.…”

Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells

“…Such sensitivity is sufficient to detect the 2500 most abundant proteins in a single human cell 18 , which should be able to identify different states of cellular differentiation. Furthermore, unlike immunoassays that require significant amounts of development time and rely on the availability of high-quality antibodies 7,8 , cPRISM-SRM is relatively easy to implement in research laboratories with commercially available standard instruments 32 .…”