DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

• Published in: Proceedings of the National Academy of Sciences - PNAS 2020-12, Vol.117 (51), p.32806-32815
• Main Authors: Karayel, Ozge, Michaelis, André C., Mann, Matthias, Schulman, Brenda A., Langlois, Christine R.
• Format: Article
• Language: English
• Subjects: Biological Sciences; Biology; Carbon - metabolism; Carbon sources; Culture Media; Fructose-Bisphosphatase - metabolism; Gene deletion; Glucose - metabolism; Glycolysis; Growth conditions; Malate Dehydrogenase - metabolism; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Metabolism; Mutation; Point Mutation; Proteomes; Proteomics; Pyruvate Decarboxylase - metabolism; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sample preparation; Scientific imaging; Sodium-Potassium-Exchanging ATPase - metabolism; Spectroscopy; Stress, Physiological; Systems Biology - methods; Target recognition; Ubiquitin; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Workflow; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2020197117

The yeast Saccharomyces cerevisiae is a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run, data-independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement, and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source-dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system readout pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation of targets of the GID E3 ligase. Moreover, this approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole-proteome–based readouts can serve as comprehensive systems-level assays in all cellular systems.