Project description:Quantitative proteomics in large cohorts is highly valuable for biomedical/pharmaceutical investigations but often suffers from suboptimal reliability, accuracy, and reproducibility. Here we describe a new ultra-high-resolution (UHR) IonStar method achieving precise/reproducible protein measurement in large-cohorts while eliminating accuracy-related problems such as ratio compression, by taking advantage of the exceptional selectivity of UHR-MS1 detection (240k_FWHM). Using mixed-proteome sets reflecting large-cohort analysis using technical or biological replicates (N=56), we comprehensively compared the quantitative performances of UHR-IonStar with a state-of-the-art SWATH-MS method. The SWATH-MS employs a cutting-edge TripleTOF and a SpectronautTM package and was meticulously optimized to maximize sensitivity, reproducibility and proteome-coverage, by developing a highly extensive, customized spectral-library, reproducible/robust capillary-LC separation and narrow, variable-window acquisition. Comparing quantitative performances of the two distinct methods (i.e. MS1-vs.MS2-based) affords interesting and valuable observations. While the performances for higher-abundance proteins (i.e. higher 75% proteins in abundance) are quite similar by the two methods, UHR-IonStar showed markedly better quantitative accuracy, precision and reproducibility (e.g. R2>0.9 by UHR-IonStar vs. R2<0.4 by SWATH-MS) for proteins of lower 25% in abundance, and much improved sensitivity/selectivity for discovering significantly-altered proteins, especially these with changes ≤2.5 folds. Furthermore, UHR-IonStar showed more accurate protein quantification in single analysis of each individual sample in a large set, which is an inadequately-investigated albeit highly critical parameter for large-cohort analysis. Finally, we compared the two methods in measuring time courses of altered proteins in cancer cells (N=36) treated by paclitaxel, where dysregulated biological processes have been well-established. UHR-IonStar discovered more altered-proteins in biological processes and pathways that are known to be induced by paclitaxel, with substantially better significance scores. Additionally, UHR-IonStar showed markedly superior ability in accurately depicting the time courses of tubulin-α/β isoforms which are well-known to be consistently induced by paclitaxel. In summary, UHR-IonStar represents a reliable, robust and cost-effective solution for large-cohort proteomics quantification with excellent accuracy and precision.

Dataset Information

Proteomic analysis of apoE-KO mice liver exposed to inhaled silicon dioxide.

Publications

Changes in the liver proteome in apoE knockout mice exposed to inhalation of silica nanoparticles indicate mitochondrial damage and impairment of ER stress responses associated with microvesicular steatosis.

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