Abstract
Metal-binding proteins (MBPs) have various and important biological roles in all living species and many human diseases are intricately linked to dysfunctional MBPs. Here, we report a chemoproteomic method named ‘metal extraction-triggered agitation logged by thermal proteome profiling’ (METAL-TPP) to globally profile MBPs in proteomes. The method involves the extraction of metals from MBPs using chelators and monitoring the resulting protein stability changes through thermal proteome profiling. Applying METAL-TPP to the human proteome with a broad-spectrum chelator, EDTA, revealed a group of proteins with reduced thermal stability that contained both previously known MBPs and currently unannotated MBP candidates. Biochemical characterization of one potential target, glutamine-fructose-6-phosphate transaminase 2 (GFPT2), showed that zinc bound the protein, inhibited its enzymatic activity and modulated the hexosamine biosynthesis pathway. METAL-TPP profiling with another chelator, TPEN, uncovered additional MBPs in proteomes. Collectively, this study developed a robust tool for proteomic discovery of MBPs and provides a rich resource for functional studies of metals in cell biology.
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Data availability
The MS proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD024925 for EDTA and PXD029856 for TPEN. Source data are provided with this paper.
Code availability
TPP data preprocessing and the fitting of melting curves were performed using custom algorithms (https://doi.org/10.5281/zenodo.10278871).
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Acknowledgements
We thank the Computing Platform of the Center for Life Science for supporting the proteomics data analysis and Analytical Instrumentation Center of College of Chemistry and Molecular Engineering, Peking University, for ICP-MS analysis. We thank the National Center for Protein Sciences at Peking University for assistance with protein crystallization and the staff of the Shanghai Synchrotron Radiation Facility and KEK Photon Factory for assistance with X-ray data collection. We thank the National Center for Protein Sciences at Peking University for assistance with the ITC experiment. We thank the Metabolomics Facility Center of Metabolomics and Lipidomics in the National Protein Science Technology Center of Tsinghua University for LC–MS/MS experiments of UDP-GlcNAc quantification. This work was supported by the National Natural Science Foundation of China (no. 21925701, no. 92153301 and no. 91953109) to C.W.
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Contributions
X.Z., W.Q. and C.W. conceptualized the project. X.Z. and X.W. conducted most of the experiments unless otherwise specified. T.W. solved the structure under the guidance of J.X. and purified GFPT1, GFPT2 and GPATCH11. Y.L. developed computational algorithms to analyze the TMT proteomic data. Z.T., Y.Z. and T.F. helped to carry out the thermal stability assay of GlmS-C, performed the activity assay of GlmS and constructed certain mutants. Y.C. and F.W. helped with data extraction and computational analysis. B.M. helped with the ICP-MS measurement. C.G. helped to refine the structure. X.Z., X.W. and C.W. analyzed the data and wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 Gel-based validation of METALL-TPP with EDTA on purified proteins and in HeLa cell lysates.
a, Four human proteins were recombinantly expressed and purified from E. coli. The experiments were repeated two times with similar results. b, The extraction of zinc from SORD by EDTA as measured by ICP-MS. c, Optimization of the EDTA concentrations to perturb SORD’s thermal stability in 2 mg/mL HeLa cell lysates by immunoblotting. GAPDH was used as a negative control. d, 4 mM EDTA treatment of HeLa cell lysates (2 mg/mL) does not affect the global proteome stability as demonstrated by SDS-PAGE stained with Coomassie Brilliant Blue (CBB). In b, error bars represent mean ± s.d. Results are from three independent experiments. Statistical differences were determined by a two-sided Student’s t-test. In c and d, the experiments were repeated two and three times with similar results, respectively.
Extended Data Fig. 2 Thermal shift curves of representative proteins quantified from the MS-based METAL-TPP profiling experiment with EDTA.
Annotated MBPs include SORD, ENO1, GSN, TRAF2 and SEPTIN2. Two novel MBP candidate proteins are RSU1 and GFPT2. GAPDH is the non-MBPs control. For each protein, the thermal shift curves were quantified from the TMT-based quantification and the Tm values were fitted by GraphPad Prism 7.0. Error bars represent mean ± s.d. Results are from three independent experiments. Statistical differences were determined by a two-sided Student’s t-test.
Extended Data Fig. 3 Analysis of proteins quantified from the METAL-TPP profiling with EDTA.
a, Analysis of the subcellular locations of those annotated MBPs found in different thermal stability groups. b, Analysis of the secondary structure content of those annotated MBPs found in different thermal stability groups. For each group, the percentage of helix, sheet and loop contents are shown. The values of each box and whisker are maximum value, upper quartile, median, lower quartile and minimum value. The cellular compartment (c) and molecular function (d) analysis of the 165 thermally stabilized proteins regardless they are annotated MBPs or not. e, Distribution of ΔTm values for annotated zinc-binding and magnesium-binding MBPs (‘Zn-MBPs’ and ‘Mg-MBPs’) quantified from the METAL-TPP profiling experiments. ΔTm values for Zn-MBPs, Mg-MBPs and all other MBPs are shown in cyan, orange and grey dots, respectively. Moving averages (window of 20) of Zn-and Mg-MBPs are shown as cyan and orange lines. All data were generated from three (n = 3) independent biological replicates. Statistical differences were determined by a two-sided Student’s t-test.
Extended Data Fig. 4 Biochemical characterization of purified GFPT1 and GFPT2.
a, SDS-PAGE of GFPT1 and GFPT2 with internal 6xHis tags that were purified from insect cells after nickel chromatography. The TEV protease (‘TEV’) was purified in parallel as a negative control. The purified proteins were further filtered to remove excessive imidazole in the buffer. b, Size exclusion chromatography (SEC) of the purified GFPT2. The protein came out as one single aggregation peak. c, Size exclusion chromatography (SEC) of the purified GFPT1. The protein came out as one aggregation peak and one active-form peak, the latter of which was resolved by SDS-PAGE. d, Measurement of metal content of the purified GFPT1 after SEC by ICP-MS. Significant zinc-binding activity was detected. In a and c, the experiment was repeated two times with similar results. In d, error bars represent mean ± s.d. Results are from three independent experiments. Statistical differences were determined by a two-sided Student’s t-test.
Extended Data Fig. 5 Biochemical characterization of recombinant GLMS as a zinc-binding protein.
a, Structures of GLMS with the two substrates bound (PDB:4AMV & PDB:1XFF). Based on these structures, glutamine (green sphere) is bound at the N-terminal domain (red ribbon) and fructose-6-P (pink sphere) was bound at the C-terminal domain (blue ribbon). b, Recombinant expression and purification of the full-length GLMS from E. coli with a GST tag. Samples were collected along the purification process and analyzed by SDS-PAGE including supernatant after cell lysis (S), flow through after GST columns (FT), three wash steps (W1, W2 and W3), elution from GST columns by 20 mM glutathione (E) and GST tag was cleaved by TEV protease (GLMS). Asterisk marks the position of purified GLMS. c, The binding affinity of zinc with GLMS was measured as 18 ± 5 μM by ITC. During measurement, 1 mM ZnSO4 (fresh) was titrated to 25 μM GLMS in 25 mM Tris buffer. d, Recombinant expression and purification of GLMS-C from E. coli with the 6xHis tag. The His tag was cleaved by TEV protease. In a and d, the experiment was repeated two times with similar results.
Extended Data Fig. 6 Testing of potential zinc-binding site on GFPT1/2 that is equivalent to C301 in GLMS.
a, GFPT2-C375A is more sensitive to zinc inhibition as compared to the wild-type (WT) GFPT2. Hela cells were transiently overexpressed with WT or C375A mutant of GFPT2 and then treated with 100 μM zinc for 30 min. After lysis, the enzymatic activity of GFPT2 were measured (n = 3). b, Zinc treatment similarly reduces the HBP flux of in both WT- and C375A-overexpressing cells. Quantitative LC-MS/MS analysis showed decreased UDP-GlcNAc levels after HeLa cells overexpressing GFPT2 WT and C375A mutant were treated with 100 μM zinc for 30 min (n = 3). c, Purified WT-GFPT1 and the C374A mutant were equally sensitive toward zinc inhibition. Recombinant WT-GFPT1 and the C374A mutant were treated with different concentration of zinc, followed by the measurement of their activity (n = 3). In a-c, error bars represent mean ± s.d. Results are from three independent experiments. Statistical differences were determined by a two-sided Student’s t-test.
Extended Data Fig. 7 Comparison of the METAL-TPP profiling results with TEPN and EDTA.
a, Overlap of MBPs with reduced thermal stability from the TPEN and EDTA datasets. b, Analysis of 37 MBPs identified both by the TPEN and EDTA profiling. c, Analysis of MBPs identified only in the TPEN dataset. d, Analysis of MBPs identified only in the EDTA dataset. All data were generated from three (n = 3) independent biological replicates.
Extended Data Fig. 8 Characterization of GPATCH11 as a zinc-binding protein.
a, EDTA treatment decreases the thermal stability of GPATCH11 in HeLa cell lysates. Thermal shift curves of GPATCH11 quantified from the MS-based METAL-TPP profiling experiment with EDTA treatment were shown. The Tm values were fitted by GraphPad Prism 7.0. Error bars represent mean ± s.d. Results are from three independent experiments. Statistical differences were determined by a two-sided Student’s t-test. b, SDS-PAGE of GPATCH11 purified from insect cells with a GST tag. Asterisk marks the position of GPATCH11-GST. The experiment was repeated two with similar results.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Supplementary Data 1
List of Tm and ΔTm values for each protein quantified from three biological replicates of METAL-TPP experiments with EDTA.
Supplementary Data 2
List of 5,833 annotated MBPs extracted from UniProt.
Supplementary Data 3
List of proteins identified with significantly reduced thermal stability by METAL-TPP with EDTA.
Supplementary Data 4
List of Tm and ΔTm values for each protein quantified from three biological replicates of METAL-TPP experiments.
Source data
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Source Data Extended Data Fig. 1
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Zeng, X., Wei, T., Wang, X. et al. Discovery of metal-binding proteins by thermal proteome profiling. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01563-y
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DOI: https://doi.org/10.1038/s41589-024-01563-y