Updated publication reference for PubMed record(s): 31444107, 33443437. In this study, we attempted to achieve to date the most comprehensive coverage of the yeast proteome during log-phase growth and in the K11R mutant ubiquitin yeast strain with a combination of (1) extensive gel molecular weight fractionation and use of either of two digestion enzymes to improve coverage by addressing the issue of dynamic range via decreased sample complexity, (2) use of a more sensitive and faster LC-MS/MS platform than available for many of the previous experiments (Orbitrap Velos coupled to a nanoAcquity UPLC). By this approach, we identified with high confidence a new fungal conserved gene expressed in S. cerevisiae and achieved the highest coverage of the yeast proteome (83.5%) to date. Besides that, we also extended our experimental workflow incorporating the same advantages to identify and quantify proteins in a K11R/reference SILAC/control total yeast lysate mixture.