Peptide Identification and Quantification by Gas Phase Fractionation Enables Proteomics Without Liquid Chromatography

Liquid chromatography mass spectrometry (LC-MS) delivers sensitive peptide analysis for proteomics, but the methodology requires extensive analysis time, hampering throughput. Here, we demonstrate that flow injection analysis data-independent acquisition (FIA-DIA), using gas-phase peptide separation instead of LC, offers extremely fast proteome analysis. Incorporating ion mobility with FIA-DIA, we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins/second). We show the utility of this technology to perform a complex multifactorial proteome study of interactions between nutrients, genotype, and mitochondrial toxins in a collection of cultured human cells. More than 45,000 quantitative protein measurements from 132 samples were achieved in only 4.4 hours of MS data collection. Enabling fast, unbiased proteome quantification without LC, FIA-DIA offers a new approach to boosting throughput critical to drug and biomarker discovery studies that require analysis of thousands of proteomes.