Unique identifications of peptides in panels A-C are the average of two independent experiments.Ĭharacterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. (E) KEGG pathway enrichment analysis of 395 proteins identified (FDR0.4. (D) Distribution of peptide log2(LFQ) values from traditional nLC-MS/MS of the same MCF7 peptides showing the subset of 1,173 peptides that were identified by the optimal FIA-DIA conditions from the scouting experiment (360 ms ion injection time, 60k resolution, 1 m/z isolation width). (C) Unique peptide identifications as a function of orbitrap resolution with maximum ion injection time fixed at 118 ms and isolation width fixed at either 2 or 6 m/z. (B) Unique peptide identifications as a function of maximum ion accumulation time with orbitrap resolution fixed at 60,000 and isolation width fixed at 1 or 2 m/z. (A) Unique peptide identifications as a function of mass filter isolation window width with resolution fixed at 60,000 and maximum ion accumulation time fixed at 118 ms. Peptide and Protein Identification by FIA-DIA improves with gas-phase fractionation. 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. More than 45,000 quantitative protein measurements from 132 samples were achieved in only 4.4 hours of MS data collection. 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. 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).
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. Liquid chromatography mass spectrometry (LC-MS) delivers sensitive peptide analysis for proteomics, but the methodology requires extensive analysis time, hampering throughput.