Shan Lu, Yong Cao, Sheng-Bo Fan, Zhen-Lin Chen, Run-Qian Fang, Si-Min He, Meng-Qiu Dong. Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures. Biophysics Reports, 2018, 4(2): 68-81. doi: 10.1007/s41048-018-0050-6
Citation: Shan Lu, Yong Cao, Sheng-Bo Fan, Zhen-Lin Chen, Run-Qian Fang, Si-Min He, Meng-Qiu Dong. Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures. Biophysics Reports, 2018, 4(2): 68-81. doi: 10.1007/s41048-018-0050-6

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

doi: 10.1007/s41048-018-0050-6
Funds:  The authors would like to thank the Beijing Municipal Science and Technology Commission,the Ministry of Science and Technology of China,and the Natural Science Foundation of China (21475141) for research funding.
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  • Corresponding author: Si-Min He, Meng-Qiu Dong
  • Received Date: 16 February 2018
  • Rev Recd Date: 08 March 2018
  • Publish Date: 30 April 2018
  • 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 fastscanning, high-resolution, and accurate-mass LC-MS system.
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