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. 2016 Apr;15(4):1381-96.
doi: 10.1074/mcp.o115.051839.

Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

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Free PMC article

Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

Yuan Gao et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.

Figures

Fig. 1.
Fig. 1.
Quantitative comparison of the binding affinity among seven different UBDs to ubiquitin and seven types of ubiquitin chains. A, flowchart for evaluating the binding affinity of recombinant UBDs to ubiquitin and ubiquitin chains with Western blot analysis and SILAC-AQUA. B, quantification of purified seven types of recombinant GST-UBDs on 10% SDS-polyacrylamide gels. A20_mut with the replacement of C35A and C38A was saved as the negative control for enrichment analysis. Same copy numbers of GST-UBDs were loaded on the gel and displayed with Coomassie Blue staining. C and D, comparison of the ubiquitin binding affinity of different UBDs. Equimolar UBDs were used to enrich ubiquitinated proteins from the same amount of yeast total cell lysates. The enriched ubiquitinated proteins were resolved by SDS-PAGE and probed with an anti-Myc antibody. The relative amount of ubiquitinated proteins was calculated based on intensity of the Western blot signal. E, quantitative comparison of the binding affinity of seven types of UBDs to seven types of ubiquitin chains. The amount of seven ubiquitin chains was measured by SILAC-AQUA. The same amount of heavy isotope-labeled ubiquitin chains purified by Ni-NTA under denaturing conditions was saved as the internal standard added to each reaction. The result was shown as relative amount compared with that from the enrichment of ubiquitinated proteins by Ni-NTA under denaturing conditions. Data are represented as mean and S.E.
Fig. 2.
Fig. 2.
Quantitative comparison of the binding affinity to ubiquitin and seven ubiquitin chains with five different types of TUBD recombinants selected from the first run of screening. A, schematic representation of tetra-TUBDs. UBD unit was linked through polyglycine linker and fused with GST on the N terminus. B, quantification of purified five types of recombinant GST-TUBDs on a 10% SDS-polyacrylamide gel. Same copy numbers of GST-TUBDs were loaded on the gel and displayed with Coomassie Blue staining. C and D, comparison of the ubiquitin binding affinity of different TUBDs. The equimolar TUBDs are used to enrich ubiquitinated proteins from the same amount of yeast total cell lysates. The enriched ubiquitinated proteins are separated by SDS-PAGE and probed with anti-Myc antibody. The relative amount of ubiquitinated proteins was calculated based on the intensity of Western blot signal. E, quantitative comparison of the binding affinity of seven types of ubiquitin chains to five types of TUBDs through SILAC-AQUA strategy as mentioned above. Same amount of heavy isotope-labeled ubiquitin chains purified by Ni-NTA under denaturing condition was saved as the internal standard added to each reaction. The result was shown as relative amount compared with that from the enrichment of ubiquitinated proteins by Ni-NTA in denature condition. Data are represented as mean and S.E.
Fig. 3.
Fig. 3.
Evaluation and optimization of ThUBD recombinant. A, quantification of purified two types of hybrid GST-hUBDs recombinant on a 10% SDS-PAGE. Dimer, trimer, and tetramer of hUBDs were recombined and fused with GST. Same copy numbers of GST-hUBDs were loaded on the gel and displayed with Coomassie Blue staining. B and C, comparison of the ubiquitin binding affinity of different types of ThUBDs with varied units. Equimolar ThUBDs were used to enrich ubiquitinated proteins from the same amount of total cell lysates. The enriched ubiquitinated proteins were separated by SDS-PAGE and probed with an anti-Myc antibody. The relative amount of ubiquitinated proteins was calculated based on the intensity of the Western blot signal. D, quantitative comparison of the binding affinity of seven types of ubiquitin chains to two types of ThUBDs with varied number of units through SILAC-AQUA strategy as mentioned above. Same amount of heavy isotope-labeled ubiquitin-chains purified by Ni-NTA under denaturing condition was saved as the internal standard added to each reaction. The result was shown as the relative amount compared with that from the enrichment of ubiquitinated proteins by Ni-NTA in denature condition. Data are represented as mean and S.E.
Fig. 4.
Fig. 4.
Characterization of ThUBD and qUBA for the enrichment of polyubiquitinated proteins and ubiquitin from yeast total cell lysates. A, flowchart for evaluation of enrichment efficiency for ubiquitinated proteins by ThUBD and qUBA with quantitative proteomics approach. B, comparison of ubiquitin conjugates purified with ThUBD and qUBA by SDS-PAGE. Ubiquitinated proteins were purified from the same amount of total cell lysates, resolved on a 10% SDS-polyacrylamide gel, stained with Coomassie Blue, excised into 18 gel bands, digested by trypsin, and analyzed by LC-MS/MS. C, comparison of the ubiquitin purified by ThUBD and qUBA in each gel band, reflecting quantitative data in all gel bands in the purified ubiquitinated proteins. The ubiquitin abundance was represented by the intensity signal on MS of Ser-57 peptide (TLSDYNIQK) extracted from each individual gel band. Data are represented as mean and S.E. D, comparison of identified ubiquitinated proteins with ubiquitin-modified sites enriched by ThUBD (n = 362) or qUBA (n = 260). E, log2 ratio distribution of the spectrum counts of identified ubiquitinated proteins with the ubiquitin-modified site enriched by ThUBD and qUBA. The ubiquitinated protein abundance was represented by the spectrum count of identified peptides extracted from each individual gel band.
Fig. 5.
Fig. 5.
Comparison of the enriched ubiquitinated proteins from yeast total cell lysates by ThUBD and Ni-NTA beads under denaturing condition (Ni_denature). A, comparison of ubiquitinated proteins purified with ThUBD and Ni-NTA beads under denaturing condition by SDS-PAGE. Ubiquitin conjugates were purified from the same amount of yeast total cell lysates, resolved on a 10% SDS-polyacrylamide gel, stained with Coomassie Blue, excised into 24 gel bands, digested by trypsin, and analyzed by LC-MS/MS. B, base-peak chromatogram of three MS runs (upper) and elution profiles of peptides Ser-57, Lys-48 (LIFAGK↓QLEDGR), and Lys-63 (TLSDYNIQK↓ESTLHLVLR) (bottom) resulting from trypsin digestion of ubiquitin or ubiquitin chains for the samples of the gel band 2 on A for total cell lysates (TCL), Ni_denature, and ThUBD, respectively. Peptides derived from the gel band 2 were loaded on the column and then eluted with a 2–30% gradient of buffer B over 50 min. The peak area (AA) corresponds to the relative ion intensity of the analyzed peptide. C, comparison of ubiquitin in total cell lysates (TCL) or purified by ThUBD and Ni-NTA under denaturing condition in each gel band, reflecting quantitative data in all gel bands in the total cell lysates or ubiquitin conjugates. The ubiquitin abundance was represented by the intensity signal on MS of Ser-57 peptide extracted from each individual gel band. Data are represented as mean and S.E. D, comparison of identified ubiquitinated proteins with ubiquitin-modified site enriched by ThUBD (n = 362), Ni_denature (this study, n = 268), and data referenced from Peng et al. (24) (n = 72). E, virtual Western blot reconstructed from proteomics data for protein ZEO1, reflecting quantitative data in all gel bands in the ubiquitin conjugates purified by ThUBD and Ni-NTA under denaturing conditions. The protein abundance was represented by the darkness and thickness of the bands, and the molecular weight information was extracted from the one-dimensional SDS gel. The arrow shows the theoretical molecular mass of ZEO1 in yeast.
Fig. 6.
Fig. 6.
Large scale profiling of ubiquitinated proteins enriched by ThUBD from liver cancer cell MHCC97-H. A, comparison of MHCC97-H total cell lysates (TCL) and ubiquitinated proteins purified by ThUBD displayed on SDS-PAGE. Both samples are resolved on a 10% SDS-polyacrylamide gel, stained with Coomassie Blue, excised into 18 gel bands, digested by trypsin, and analyzed by LC-MS/MS. B, base-peak chromatogram of two MS runs (upper) and elution profiles of peptides Ser-57, Lys-48, and Lys-63 (bottom) resulting from trypsin digestion of ubiquitin or ubiquitin chains for the samples of the gel band 3 on A for total cell lysates and ubiquitinated proteins (ubc), respectively. Peptides derived from the gel band 3 were loaded on the column and then eluted in a 2–30% gradient of buffer B over 50 min. The peak area (AA) corresponds to the relative ion intensity of the analyzed peptide. C, ΔMW (the molecular weight difference between the experimental and theoretical one) histogram of the proteins identified in the total cell lysates (n = 4518) and ubiquitinated proteins (ubc) (n = 2551). The dataset was produced through SDS-PAGE separation and then analyzed by LC-MS/MS. D, comparison of ubiquitinated proteins purified by ThUBD, qUBA, and TUBE from mammalian cells.
Fig. 7.
Fig. 7.
pH RP-LC separation efficiently increases the identification of ubiquitinated proteins and ubiquitination sites, compared with SDS-polyacrylamide gel separation. A, comparison of the characteristics of gel and pH RP-LC separation for the ubiquitin conjugates' identification through LC-MS/MS analysis. B and C, distribution of the certain peptide from ubiquitin (B) or HSPA8 (C) in different fractions separated by gel and pH RP-LC. The K# in the HSPA8 peptide stands for ubiquitin modified on the lysine. D and E, identified potential ubiquitinated proteins and ubiquitinated proteins with ubiquitin-modified sites separated by pH-RP-LC and SDS-polyacrylamide gel, respectively. All proteins are converted to UniProtKB ID for comparison. F, top 15 pathways integrate the ubiquitinated proteins with ubiquitin-modified sites purified by ThUBD (n = 1125) from MHCC97-H cells. G, amino acid sequence properties of ubiquitination sites enriched in MHCC97-H cells and yeast, respectively. H, top 10 pathways enriched by the conserved proteins (n = 305) purified from MHCC97-H cells and yeast.

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