Significance 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 comprehensive resource detailing changes to the yeast proteome following commonly used stress treatments in yeast. Our systems biology approach identifies and validates regulatory targets of an E3 ubiquitin ligase during a metabolic switch, providing insights into the interplay of metabolic pathways. The speed, simplicity, and scalability of this workflow makes it particularly well-suited for screens in any cellular system to investigate specific effects of deletions or mutants or other perturbations to obtain the response of biological system on a global level.