Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation

蛋白质组 计算生物学 蛋白质组学 转录组 单细胞分析 质谱法 细胞周期蛋白 细胞周期 染色质 化学 系统生物学 细胞 稳健性(进化) 生物 细胞生物学 生物信息学 DNA 生物化学 色谱法 基因表达 基因
作者
Andreas-David Brunner,Marvin Thielert,Catherine G. Vasilopoulou,Constantin Ammar,Fabian Coscia,Andreas Mund,Ole Hørning,Nicolai Bache,Amalia Apalategui,Markus Lübeck,Oliver Raether,Melvin Park,Sabrina Richter,David Fischer,Fabian J. Theis,Florian Meier,Matthias Mann
标识
DOI:10.1101/2020.12.22.423933
摘要

Abstract Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing 1–3 . However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary 4,5 . Chemical labeling-based single-cell approaches introduce hundreds of cells into the MS, but direct analysis of single cells has not yet reached the necessary sensitivity, robustness and quantitative accuracy to answer biological questions 6,7 . Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography and a novel trapped ion mobility mass spectrometer, resulting in a more than ten-fold improved sensitivity. We accurately and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators such as CDK2NA, the E2 ubiquitin ligase UBE2S, DNA topoisomerases TOP2A/B and the chromatin regulator HMGA1. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable core proteome despite perturbation, while the transcriptome appears volatile. This emphasizes substantial regulation of translation and sets the stage for its elucidation at the single cell level. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material 8 , posttranslational modifications and small molecule studies to gain unprecedented insights into cellular heterogeneity in health and disease.
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