Quantitative shotgun proteome analysis by direct infusion

Liquid chromatography–mass spectrometry (LC–MS) delivers sensitive peptide analysis for proteomics but requires extensive analysis time, reducing throughput. Here, we demonstrate that gas-phase peptide separation instead of LC enables fast proteome analysis. Using direct infusion–shotgun proteome analysis (DI-SPA) by data-independent acquisition mass spectrometry (DIA-MS), we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins per second). We show the utility of this technology in performing a complex multifactorial proteomic 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 h of MS data collection. Enabling fast, unbiased proteome quantification without LC, DI-SPA offers an approach to boost throughput, critical to drug and biomarker discovery studies that require analysis of thousands of proteomes.