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Detecting native folds in mixtures of proteins that contain disulfide bonds

Nature Biotechnology volume 26, pages 427–429 (2008)Cite this article

Abstract

High-throughput in vitro refolding of proteins that contain disulfide bonds, for which soluble expression is particularly difficult, is severely impeded by the absence of effective methods for detecting their native forms. We demonstrate such a method, which combines mass spectrometry with mild reductions, requires no prior experimentation or knowledge of proteins' physicochemical characteristics, function or activity, and is amenable to automation. These are necessary criteria for structural genomics and proteomics applications.

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Figure 1: ESI/FTMS mass spectra of seven multiple-disulfide-bond–containing proteins (a) The regeneration mixture, 14 h after initiation of oxidative folding (50 mM DTTox, 10 mM Ca2+, 20 mM Tris-HCl, pH 8) that was subjected to a reduction pulse, followed by AEMTS blocking.

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Acknowledgements

This research was supported by National Institutes of Health grants GM-24893 (to H.A.S.) and GM-16609 (to F.W.M.), by NORT(DNT), Hungary, OTKA-NF61431 (to E.W.), and by UTEP startup money (to M.N.). E.W. is a Howard Hughes Medical Institute international scholar and an EMBO-HHMI start up grantee.

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Author notes

  1. Mahesh Narayan and Ervin Welker: These authors contributed equally to this work.

Authors and Affiliations

  1. Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, 14353-1301, New York, USA

    Mahesh Narayan, Ervin Welker, Huili Zhai, Xuemei Han, Guoqiang Xu, Fred W McLafferty & Harold A Scheraga

  2. Department of Chemistry, University of Texas at El Paso, El Paso, Texas, 79968, USA

    Mahesh Narayan

  3. Institute of Biochemistry, Biological Research Centre of the Hungarian Academy, H-6701, Szeged, 62 Temesvari krt., Hungary

    Ervin Welker

  4. Institute of Enzymology, Biological Research Centre of the Hungarian Academy, H-1114, Budapest, 29 Karolina ut., Hungary

    Ervin Welker

Authors
  1. Mahesh Narayan
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  2. Ervin Welker
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  3. Huili Zhai
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  4. Xuemei Han
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  5. Guoqiang Xu
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  6. Fred W McLafferty
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  7. Harold A Scheraga
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Corresponding authors

Correspondence to Mahesh Narayan, Ervin Welker or Harold A Scheraga.

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Supplementary Tables 1,2, Notes, Methods (PDF 189 kb)

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Narayan, M., Welker, E., Zhai, H. et al. Detecting native folds in mixtures of proteins that contain disulfide bonds. Nat Biotechnol 26, 427–429 (2008). https://doi.org/10.1038/nbt1380

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