Figure 2.

Mutant TPP workflow adaptations. A, equal amounts of protein from each lysate were subjected to six different temperature treatments, 35.0, 45.3, 50.1, 55.2, 60.7, and 74.9 °C, to induce protein denaturation. The soluble fractions from each treatment were digested in-solution with trypsin/Lys-C. The resulting peptides were labeled with isobaric mass tags (TMTsixplex™) and mixed by genotype prior to MS analysis. Resulting MS/MS data were analyzed using Proteome Discoverer™ 2.2 to identify and quantify abundance levels of peptides for each temperature treatment and each genotype. B, dot plots showing the abundance value for every protein detected from the MTPP experiments in WT, rpn5-ts, and pup2-ts. Box-and-whisker plots include minimum, maximum, and median. C, representative melt curves from a single replicate of the percentage of soluble protein following heat treatment are shown for selected protein complexes. Proteins isolated from WT are shown in gray, rpn5-ts in orange, and pup2-ts in teal. Shown here are curves of the RNA exosome (11 individual proteins), the 40S ribosome (39 individual proteins), and the chaperonin-containing T-complex (8 individual proteins). D, individual protein melt curves were created for every quantified protein and normalized using the TPP R package. T_m was calculated as the temperature at which 50% of the protein was denatured. Shown are one protein from each of the complexes in B.