A new reagent for the routine identification of cysteine residues during protein sequencing is described. This method employs 3-bromopropylamine to alkylate cysteines in proteins after reduction with dithiothreitol. Upon sequencing of the protein on an Applied Biosystems 477A protein sequencer, the aminopropylcysteine residue yields a phenylthiohydantoin (PTH) derivative which elutes reproducibly at a unique position immediately after PTH-leucine; baseline resolution is achieved without modification of the PTH analyzer gradient. Unlike PTH-pyridylethylcysteine, the relative elution position of the aminopropylcysteine PTH derivative is not affected by changes in the ionic strength of the analyzer solvents. In addition, the previewing of the next amino acid which is observed in proteins modified with 4-vinylpyridine does not occur in aminopropylated proteins. Preparation of alkylated proteins for electroblotting onto polyvinylidene difluoride (PVDF) membranes and methods for desalting alkylated proteins immobilized on precoated glass fiber filters or PVDF membranes are also described.

中文翻译：

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通过蛋白质序列分析鉴定用3-溴丙胺烷基化的半胱氨酸残基。

描述了一种在蛋白质测序过程中常规鉴定半胱氨酸残基的新试剂。该方法在用二硫苏糖醇还原后，使用3-溴丙胺对蛋白质中的半胱氨酸进行烷基化。在Applied Biosystems 477A蛋白质测序仪上对蛋白质进行测序后，氨丙基半胱氨酸残基产生苯硫基乙内酰脲（PTH）衍生物，该衍生物可重复性地在PTH-亮氨酸后的唯一位置洗脱；无需更改PTH分析仪梯度即可实现基线分辨率。与PTH-吡啶基乙基半胱氨酸不同，氨丙基半胱氨酸PTH衍生物的相对洗脱位置不受分析仪溶剂离子强度的变化影响。此外，在用4-乙烯基吡啶修饰的蛋白质中观察到的下一个氨基酸的预演在氨基丙基化的蛋白质中不会发生。还描述了用于电印迹到聚偏二氟乙烯（PVDF）膜上的烷基化蛋白的制备以及使固定在预涂玻璃纤维滤膜或PVDF膜上的烷基化蛋白脱盐的方法。