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

ABSTRACT: Yeast Saccharomyces cerevisiae is a powerful model system for systems-wide biology screens and large-scale proteomics methods. The proteomics community has achieved nearly complete coverage for this organism owing to advances in mass spectrometry. However, it remains challenging to scale the 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 approach 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 largely covered yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to a variety of stresses commonly used in yeast research, identify distinct responses to each stress, and provide a comprehensive resource of these responses. Using our facile, rapid and robust methodology, we observe many previously characterized stress responses, including carbon source dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. Furthermore, we applied our methodology to search for new regulatory targets of the GID ligase during a metabolic switch. We are able to pinpoint effects of a single deletion or point mutation in the GID complex on the global proteome, and thereby identify and validate 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Mario Oroshi  

LAB HEAD: Matthias Mann

PROVIDER: PXD021559 | Pride | 2020-11-26

REPOSITORIES: Pride