Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.
Jnana Therapeutics, Boston, Massachusetts 02210, United States.
J Am Chem Soc. 2021 May 5;143(17):6691-6700. doi: 10.1021/jacs.1c02509. Epub 2021 Apr 20.
Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.
重氮甲烷广泛用于光亲和标记 (PAL) 以捕获与生物分子的非共价相互作用。然而，如果没有对重氮甲烷与蛋白质生物分子反应性的分子理解，设计和解释 PAL 实验是具有挑战性的。在此，我们报告了对烷基和芳基重氮甲烷与单个氨基酸、单个蛋白质和整个细胞蛋白质组的标记偏好的系统评估。我们发现，烷基重氮甲烷以依赖 pH 的方式优先标记酸性氨基酸，这是反应性烷基重氮中间体的特征，而芳基-氟代重氮甲烷的标记模式反映了主要通过卡宾中间体的反应。从对 32 种烷基重氮甲烷探针的调查中，我们使用这种反应性特征来解释为什么烷基重氮甲烷探针优先富集高度酸性的蛋白质或那些嵌入膜中的蛋白质，以及为什么带有净正电荷的探针在细胞和体外往往会产生更高的标记产率。这些结果表明，烷基重氮甲烷是一种特别有效的膜蛋白质组调查化学方法，并将有助于设计和解释重氮甲烷的生物分子标记实验。
