DIA-Based Systems Biology Approach Unveils E3 Ubiquitin Ligase-Dependent Responses to a Metabolic Shift
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DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift Ozge Karayela,1, André C. Michaelisa, Matthias Manna,2, Brenda A. Schulmanb,2, and Christine R. Langloisb,1,2 aDepartment of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and bDepartment of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Contributed by Brenda A. Schulman, November 8, 2020 (sent for review September 28, 2020; reviewed by Angus I. Lamond and Alexander Varshavsky) The yeast Saccharomyces cerevisiae is a powerful model system (9), and the recently developed SWAp-tag library (10–12), has for systems-wide biology screens and large-scale proteomics meth- made yeast a premier model system for conducting transcriptomics, ods. Nearly complete proteomics coverage has been achieved owing proteomics, interactomics, or metabolomics screens (13–19). In- to advances in mass spectrometry. However, it remains challenging to deed, systems-wide biology screens and large-scale proteomics were scale this technology for rapid and high-throughput analysis of the both pioneered in the yeast model. Furthermore, the cellular in- yeast proteome to investigate biological pathways on a global scale. teraction networks and molecular mechanisms ascertained in yeast Here we describe a systems biology workflow employing plate-based can be readily applied to other systems (20–22). sample preparation and rapid, single-run, data-independent mass Early genome-wide studies showed that over 4,000 proteins are spectrometry analysis (DIA). Our approach is straightforward, easy expressed during log-phase growth in yeast and this organism was to implement, and enables quantitative profiling and comparisons the first whose entire proteome was mapped by mass spectrometry of hundreds of nearly complete yeast proteomes in only a few days. (MS)-based proteomics (23). Subsequently, yeast has served as a We evaluate its capability by characterizing changes in the yeast pro- model of choice for the development of ever-more-sensitive and teome in response to environmental perturbations, identifying dis- faster proteomics workflows (23–34). Remarkably, the optimized tinct responses to each of them and providing a comprehensive sample preparation coupled with MS analysis performed on the resource of these responses. Apart from rapidly recapitulating previ- Orbitrap hybrid mass spectrometer allowed identification of SYSTEMS BIOLOGY ously observed responses, we characterized carbon source-dependent around 4,000 yeast proteins over a 70-min liquid chromatography regulation of the GID E3 ligase, an important regulator of cellular (LC)-MS/MS run (24, 30). However, the necessity of technological metabolism during the switch between gluconeogenic and glycolytic expertise and lengthy analysis times for high-quality, in-depth growth conditions. This unveiled regulatory targets of the GID ligase yeast proteome measurements has so far precluded the wide- during a metabolic switch. Our comprehensive yeast system readout spread adoption of cutting-edge proteomics workflows in the yeast pinpointed effects of a single deletion or point mutation in the GID research community. With further advances in technology and complex on the global proteome, allowing the identification and val- new acquisition modes, such as data-independent acquisition idation of targets of the GID E3 ligase. Moreover, this approach (DIA) (35, 36), we hypothesized that it would now be possible to allowed the identification of targets from multiple cellular pathways obtain accurate and high yeast proteome coverage by a straight- that display distinct patterns of regulation. Although developed in forward and rapid single-run approach, enabling researchers to yeast, rapid whole-proteome–based readouts can serve as compre- hensive systems-level assays in all cellular systems. Significance yeast systems biology | mass spectrometry | proteomics | stress | GID E3 ligase We use a single-run, data-independent acquisition–based mass spectrometry approach to generate and compare dozens of yeast proteomes in less than a day, and provide a compre- roteome remodeling has repeatedly proven to be a vital hensive resource detailing changes to the yeast proteome fol- cellular mechanism in response to stress, changes in envi- P lowing commonly used stress treatments in yeast. Our systems ronmental conditions, and toxins or pathogens. Cells must both biology approach identifies and validates regulatory targets of synthesize proteins which enable them to adapt to the new envi- an E3 ubiquitin ligase during a metabolic switch, providing ronmental condition and inactivate or degrade proteins which are insights into the interplay of metabolic pathways. The speed, detrimental or no longer needed. For each environmental simplicity, and scalability of this workflow makes it particularly perturbation, the proteome must be precisely and distinctly well-suited for screens in any cellular system to investigate remodeled to ensure healthy and viable cells (1). Indeed, de- specific effects of deletions or mutants or other perturbations creases in proteome integrity are hallmarks of many human to obtain the response of biological system on a global level. diseases, including cancer, Alzheimer’s disease, muscular dys- – trophies, and cystic fibrosis (2 4). Despite the importance of Author contributions: O.K., M.M., B.A.S., and C.R.L. designed research; O.K. and C.R.L. cellular stress responses, our understanding of how cellular performed research; O.K., A.C.M., and C.R.L. contributed new reagents/analytic tools; O.K. pathways interact during adaptation remains incomplete. and C.R.L. analyzed data; and O.K., M.M., B.A.S., and C.R.L. wrote the paper. Therefore, knowing precisely how the proteome changes at a Reviewers: A.I.L., University of Dundee; and A.V., California Institute of Technology. global level in response to environmental cues is crucial for The authors declare no competing interest. identifying the underlying molecular mechanisms that facilitate This open access article is distributed under Creative Commons Attribution-NonCommercial- cellular adaptation. NoDerivatives License 4.0 (CC BY-NC-ND). The yeast Saccharomyces cerevisiae is a powerful model system 1O.K. and C.R.L. contributed equally to this work. that is widely used to probe biological pathways, due to its ease 2To whom correspondence may be addressed. Email: [email protected], of manipulation and rapid growth compared to mammalian [email protected], or [email protected]. models. In addition, the availability of extensive genetic re- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sources in yeast, including deletion libraries (5, 6), green fluo- doi:10.1073/pnas.2020197117/-/DCSupplemental. rescent protein–tagged libraries (7, 8), overexpression libraries www.pnas.org/cgi/doi/10.1073/pnas.2020197117 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 easily study biological processes on a global scale. Such a system Combined with our own comprehensive spectral library, the could then serve as a template for more complex proteomes, in- 23-min DIA method on average identified 33,909 peptides and cluding the human proteome. 3,413 distinct proteins in single measurements of six replicates One mechanism of maintaining proteome integrity is the (Q-value less than 1% at protein and precursor levels; Fig. 1 B marking and degradation of proteins that are damaged or no and C). This implies that ∼73% of proteins in the deep yeast longer needed with ubiquitin. The conjugation of ubiquitin to its spectral library were matched into the single runs. Note that the targets is catalyzed by E3 ubiquitin ligases, a diverse group of single runs represent only yeast grown in rich media (yeast extract enzymes that recognize and bind target proteins and facilitate peptone dextrose [YPD]), whereas the library combines the pro- ubiquitin transfer together with an E2, ubiquitin-conjugating teomes of yeast grown under several growth conditions and enzyme. Ubiquitination relies on a variety of cellular signals to therefore contains proteins which are not expressed during growth direct E3 ligases to their target proteins, and tight regulation of in YPD. Therefore, the degree of proteome completeness is likely this process is crucial for cellular viability (37). For instance, much higher than 73%. Measurements were highly reproducible during carbon starvation, yeast cells induce expression of the with Pearson coefficients greater than 0.92 between replicates (SI inactive GID (glucose-induced degradation) E3 ligase, which is Appendix,Fig.S1A) and coefficients of variation <20% for 68% of subsequently activated upon glucose replenishment. Following all common proteins between the six replicates. In comparison, a its activation, the GID E3 ligase targets gluconeogenic enzymes, single-run, data-dependent acquisition strategy with the same LC leading to their degradation and sparing the yeast from ener- gradient quantified only 11,883 peptides and 2,289 distinct pro- getically costly metabolic pathways that are unnecessary in fer- teins on average (Fig. 1 B and C). To more directly compare the mentable carbon sources (38–40). In addition, ubiquitin ligases performance of the 23-min DIA method to the DDA method we also serve as crucial regulators in response to oxidative, heavy analyzed the same sample with increasing