We report the identification of a photocleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo), which can be rapidly degraded by ultraviolet irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to that of sodium dodecyl sulfate (SDS) and is compatible with mass spectrometry. Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Moreover, Azo is simple to synthesize and can be used as a general SDS replacement in SDS–polyacrylamide gel electrophoresis.
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All data generated or analyzed during this study are presented in this article or in the provided supplementary materials. Raw gel, blot and mass spectra data are available as Supplementary Data, and source data for Fig. 1 and Supplementary Figs. 4, 9, 10 and 13 are available online. Proteomics data have been uploaded to the PRIDE repository via ProteomeXchange with identifier PXD010825.
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This research is supported by National Institutes of Health R01 GM117058 (to S.J. and Y.G.). Y.G. acknowledges R01 HL109810, R01 HL096971, R01 GM125085 and S10 OD018475. MaSDeS was a gift from S. Saveliev (Promega Corporation). We thank A. Chen, E. Chang and W. Tang for their assistance in the early stage of the project, S. Mitchell and T. Tucholski for the help with graphics, and T. Hacker for providing the swine hearts. We thank M. Willetts at Bruker for his assistance with DataAnalysis software. We also acknowledge A. Carr, E. Bayne and J. Melby for their help testing the Supplementary Protocol to ensure reproducible results.
The University of Wisconsin–Madison has filed a provisional patent application P180335US01, US serial number 62/682027 (7 June 2018) on the basis of this work. Y.G., S.J., K.B. and T.M.G.-A. are named as inventors on the provisional patent application.
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Supplementary Figs. 1–19, Supplementary Table 1 and Supplementary Notes 1–6
Detected proteoforms from LC–MS/MS run 1 (cardiac tissue)
Total detected proteoforms in 3 LC–MS/MS experiments: run 1 (cardiac tissue), run 2 (sarcoplasmic reticulum (SR) and mitochondria (Mit) enriched cardiac tissue), and run 3 (SR and Mit enriched cardiac tissue)
Proteoforms identified by TopPIC from cardiac tissue using Azo from all three LC–MS/MS runs in this study
Proteins identified by TopPIC from cardiac tissue using Azo from all three LC–MS/MS runs in this study
Subunits of the electron transport chain identified from cardiac tissue samples using Azo in this study
Integral membrane proteins identified from cardiac tissue samples using Azo in this study
ATP synthase subunits identified from cardiac tissue using Azo in this study
TopPIC proteoform identification: Azo protein extraction of human embryonic kidney (HEK) cell
TopPIC protein identification: Azo protein extraction of human embryonic kidney (HEK) cell
Full gels, blots and raw mass spectra
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Brown, K.A., Chen, B., Guardado-Alvarez, T.M. et al. A photocleavable surfactant for top-down proteomics. Nat Methods 16, 417–420 (2019). https://doi.org/10.1038/s41592-019-0391-1
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