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

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 new regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system read-out pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation novel targets of the GID E3 ligase. Moreover, our 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 assay in all cellular systems.