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Protocol | Open Access

Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures

Shan Lu1, Yong Cao1, Sheng-Bo Fan2,3, Zhen-Lin Chen2,3, Run-Qian Fang2,3, Si-Min He2,3( ), Meng-Qiu Dong1( )
National Institute of Biological Sciences, Beijing, Beijing 102206, China
Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), University of CAS, Institute of Computing Technology, CAS, Beijing 100190, China
University of Chinese Academy of Sciences, Beijing 100049, China
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Abstract

Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography–mass spectrometry (LC–MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples—purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow—from sample preparation to LC–MS and data analysis—is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC–MS system.

Keywords

Mass spectrometryCross-linkingDisulfideIdentification of disulfide bondsPLinkPLink-SS

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Biophysics Reports
Volume 4 Issue 2,
April 2018
Pages 68-81
DOI: 10.1007/s41048-018-0050-6
Cite this article:
Lu S, Cao Y, Fan S-B, et al. Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures. Biophysics Reports, 2018, 4(2): 68-81. https://doi.org/10.1007/s41048-018-0050-6

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Received: 16 February 2018
Accepted: 08 March 2018
Published: 23 April 2018
© The Author(s) 2018

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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