McDonald Lucy, Beynon Robert J
Proteomics and Functional Genomics Group, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool L69 7ZJ, UK.
Nat Protoc. 2006;1(4):1790-8. doi: 10.1038/nprot.2006.317.
We describe a protocol for selective extraction of the amino (N)-terminal-most peptide of a protein or a mixture of proteins after proteolysis. The first stage of the protocol blocks the free amino groups alpha and epsilon (the latter being lysyl residues) on the intact proteins by acetylation. In the second stage, proteolysis of the acetylated proteins yields a mixture of N-terminally acetylated (true N-terminal) and non-acetylated (internal and carboxy-terminal) peptides. Affinity capture of peptides bearing free amino groups using an immobilized amine-reactive reagent removes internal peptides from the mixture. The unbound fraction is highly enriched in N-terminal peptides, which can be analyzed without further treatment. This method is compatible with a range of proteolytic enzymes and fragmentation methods, and should take 2 d to complete. The N-terminal peptides can then be analyzed by mass spectrometry. This low cost, rapid method is readily adopted using off the shelf reagents.
我们描述了一种在蛋白质水解后选择性提取蛋白质或蛋白质混合物中最靠近氨基（N）端肽段的方法。该方法的第一阶段通过乙酰化封闭完整蛋白质上的游离α氨基和ε氨基（后者为赖氨酰残基）。在第二阶段，乙酰化蛋白质的水解产生N端乙酰化（真正的N端）肽段和非乙酰化（内部和羧基端）肽段的混合物。使用固定化的胺反应试剂对带有游离氨基的肽段进行亲和捕获，可从混合物中去除内部肽段。未结合部分富含N端肽段，无需进一步处理即可进行分析。该方法与多种蛋白水解酶和片段化方法兼容，完成整个过程大约需要2天时间。然后可以通过质谱对N端肽段进行分析。这种低成本、快速的方法使用现成的试剂即可轻松实现。
