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.
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Marta
M
Isasa
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Christopher M
CM
Rose
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Suzanne
S
Elsasser
National Autonomous University of Mexico, Av. Universidad 3000, Mexico City, District Federal 04510, Mexico.
José
J
Navarrete-Perea
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Joao A
JA
Paulo
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Daniel J
DJ
Finley
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Steven P
SP
Gygi
D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005800:Genes, Fungal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D040081:Protein Array Analysis; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; D010710:Phosphates; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D003576:Electron Transport Complex IV; D010450:Endopeptidases; C000592929:Otu1 protein, S cerevisiae; C403282:UBP10 protein, S cerevisiae; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; C101111:UBP3 protein, S cerevisiae; C561120:Ubp15 protein, S cerevisiae
COX complex; Orbitrap Fusion; TMT; UBP3; deubiquitinases; high-throughput proteomics; inorganic phosphate pathway; isobaric labeling; quantitative proteomics; ubiquitin