Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains

Characterizing a protein’s
function often requires a description
of the cellular state in its absence. Multiplexing in mass spectrometry-based
proteomics has now achieved the ability to globally measure protein
expression levels in yeast from 10 cell states simultaneously. We
applied this approach to quantify expression differences in wild type
and nine deubiquitylating enzyme (DUB) knockout strains with the goal
of creating “information networks” that might provide
deeper, mechanistic insights into a protein’s biological role.
In total, more than 3700 proteins were quantified with high reproducibility
across three biological replicates (30 samples in all). DUB mutants
demonstrated different proteomics profiles, consistent with distinct
roles for each family member. These included differences in total
ubiquitin levels and specific chain linkages. Moreover, specific expression
changes suggested novel functions for several DUB family members.
For instance, the ubp3Δ mutant showed large
expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial
regulation. Several DUBs also showed broad expression changes for
phosphate transporters as well as other components of the inorganic
phosphate signaling pathway, suggesting a role for these DUBs in regulating
phosphate metabolism. These data highlight the potential of multiplexed
proteome-wide analyses for biological investigation and provide a
framework for further study of the DUB family. Our methods are readily
applicable to the entire collection of yeast deletion mutants and
may help facilitate systematic analysis of yeast and other organisms.