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

• 来源期刊: 生物物理学报
• 年，卷（期）: 2018,（2）
• 页码范围: 68-81
• 页数: 14页
• 语种: 中文

摘要

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.