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

Biophysical Reports
Shan LuMeng-Qiu Dong

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.

References

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Citations

Sep 29, 2018·The Journal of Biological Chemistry·Ole Jørgen KaasbøllHåvard Attramadal
Apr 17, 2020·Frontiers in Immunology·Jian-Min LvBin Cheng
Dec 2, 2020·Proceedings of the National Academy of Sciences of the United States of America·Yoshihisa SuzukiJoseph Schlessinger
Nov 26, 2019·Molecular Immunology·Naeem UllahHai-Yun Li
May 25, 2020·Journal of Structural Biology·M L GiglioH Heras
Nov 3, 2020·Journal of the American Society for Mass Spectrometry·Xiaoyue Yang, Yu Xia

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Methods Mentioned

BETA
electrophoresis
X-ray
nuclear magnetic resonance
protein assay
glycosylation

Software Mentioned

pParse
Python
NET
framework
Peaks
Windows
Exactive
pFind
MSFileReader
openpyxl

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