Fig 1.
Measurement of proteasome activity in Q7Q7, Q7Q111 and Q111Q111 cells.
Cell extracts were incubated with fluorogenic peptide substrates for 1 hour at 37°C, following which enzyme activity was determined by fluorescence measurement of AMC. The activity in Q7Q111 and Q111Q111 cells is expressed as percent enzyme activity relative to the Q7Q7 cells. Activities of the chymotrypsin-like (β5) and trypsin-like (β1) proteolytic subunits were measured by cleavage of Suc-Leu-Leu-Val-Tyr-AMC (A) and Ac-Arg-Leu-Arg-AMC (B) respectively. The error bars show standard error of mean (n = 10 for Suc-Leu-Leu-Val-Tyr-AMC and n = 5 for Ac-Arg-Leu-Arg-AMC). Statistical analysis was performed using Student’s t-test: *, p ≤ 0.05; ns, no significant difference (p > 0.05) versus Q7Q7 cells.
Fig 2.
Representative MS data from peptidomics experiment comparing peptide levels between Q7Q7, Q7Q111 and Q111Q111 cells.
In this experiment, the two replicates of Q7Q7 cells were labeled with D0- and D3-TMAB-NHS, one replicate of the Q7Q111 cells was labeled with D9-TMAB-NHS and two replicates of Q111Q111 cells were labeled with D6- and D12-TMAB-NHS. A: Example of a peptide that is present in the Q111-expressing cell lines at levels much lower than in Q7Q7 cells. This peptide was identified by MS/MS analysis as TLLIKTVETRDGQVINETSQHHDDLE, derived from vimentin. B: Example of a peptide that is present in the Q111-expressing cell lines at levels slightly lower than in Q7Q7 cells. This peptide was identified by MS/MS analysis as Ac-MDTSRVQPIKLAR, derived from the 40S ribosomal protein S28. C: Example of a peptide that is present in all cell lines at comparable levels. This peptide was identified by MS/MS analysis as Ac-ADEIAKAQVAQPGGDTIFGKIIRK, derived from histidine triad nucleotide-binding protein 1.
Fig 3.
Summary plots of the peptidome of Q7Q7, Q7Q111 and Q111Q111 cells.
The relative levels of all peptides identified by MS/MS analysis in the Q7Q111 (A) and Q111Q111 (B) cells were compared to the average level of the peptide in Q7Q7 cells. The y-axis represents relative peptide levels (log-scale) and the x-axis represents the rank order of peptides sorted according to the relative level. For those peptides detected multiple times (different charge states and/or tag numbers), the relative ratio for each form was considered separately. If the ratio was <0.20, the value was capped at 0.20 to reflect the typical signal to noise ratio. The red circles represent the ratio of each replicate of the identified peptides in Q7Q111 or Q111Q111 cells, expressed relative to the level in Q7Q7 cells for that LC-MS run. The black circles represent the ratio of each Q7Q7 replicate expressed relative to the average level of Q7Q7 replicates in that LC-MS run (i.e. run 1 and 2; the other two runs did not include replicates of Q7Q7 cells).
Fig 4.
Summary plots of the peptidome of Q7Q7 cells in response to proteasome inhibitors.
A: Cells were treated with 200 nM epoxomicin for 1 hour. B: Cells were treated with 500 nM bortezomib for 1 hour. The relative levels of all peptides identified by MS/MS analysis in each of the inhibitor-treated replicates was compared to the average level of the peptide in the untreated control replicates. The y-axis represents the relative peptide levels (log-scale) and the x-axis represents the rank order of peptides sorted according to the relative level. If the ratio was <0.20 or >5.0, the value was capped at 0.20 or 5.0 to reflect the typical signal to noise ratio. Red circles indicate the ratio of each replicate of identified peptides in inhibitor-treated cells, expressed relative to the average control value. Black circles indicate the ratio of each control replicate expressed relative to the average control value.
Fig 5.
Heat map analysis of selected peptides.
Peptides frequently observed in multiple experiments were selected for this analysis. Each row denotes a specific peptide, each column represents a different experiment and each sub-column indicates peptide levels in the biological replicates within that experiment. Ratios of peptides found in multiple charged states and with different tag numbers were averaged together. The ratio was color-coded using the scheme shown at the bottom of the figure, with green representing decreases and red representing increases. Grey represents peptides that did not change substantially. White corresponds to peptides that were either not detected or which could not be accurately quantified due to peak overlap with another co-eluting peptide. Names of proteins, peptide sequences, and peptide ratios are included in S2 Table.
Table 1.
Peptides commonly detected in Q7Q7 cells and their relative levels in Q111-expressing cells or proteasome inhibitor treated cells, compared to levels in untreated Q7Q7 cells.
Fig 6.
Analysis of amino acids in the P1 position of the cleavage site.
All identified peptides in each experiment were grouped into categories based on levels relative to those in untreated Q7Q7 cells. Decrease (green bars) represent ratio ≤0.80, no change (grey bars) represent ratios of 0.81 to 1.24, increase (red bars) represent ratio ≥1.25. The residue in the P1 position of the cleavage site was considered for every peptide; peptides found in multiple replicates were counted each time found. For peptides that represent the N-terminal or C-terminal fragment of the protein, a single cleavage is sufficient to generate the peptide. Peptides that represent internal fragments of proteins require two cleavages, and so the number of cleavage sites is greater than the number of peptides. A: Cleavage site analysis of peptides in Q111-expressing cells. The data for peptides in heterozygous Q7Q111 cells were pooled with the data for peptides in homozygous Q111Q111 cells, resulting in 1441 cleavage sites for peptides that decreased (relative to Q7Q7 cells) and 73 cleavage sites for peptides in the “no change” group. Only 12 cleavage sites from 7 peptides were in the “increased” group, so it was not included in the graph. B: Cleavage site analysis of peptides in Q7Q7 cells treated with 200 nM epoxomicin for 1 hour. Peptides found to decrease represent 775 cleavage sites, the “no change” group represent 82 cleavage sites, and the increase group represents 51 cleavage sites. C: Cleavage site analysis of peptides in Q7Q7 cells treated with 500 nM bortezomib for 1 hour. There were 417 cleavage sites in the group that decreased, 59 cleavage sites in the unchanged group, and 183 cleavage sites in the group that increased after the treatment.