Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine
Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine
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DOI：
10.1007/s13361-014-0944-6
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发表时间：
2014-07
影响因子：
3.2
通讯作者：
Jens Pettelkau;C. Ihling;P. Frohberg;L. Werven;O. Jahn;A. Sinz
中科院分区：
文献类型：
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作者：
Jens Pettelkau;C. Ihling;P. Frohberg;L. Werven;O. Jahn;A. Sinz
我们描述了使用13 C标记的人工氨基酸对苯甲酰基-L-苯丙氨酸(Bpa)来提高交联产物鉴定的可靠性。我们的策略以两种蛋白质-肽复合物为例。这些研究表明，在许多情况下，在没有额外稳定同位素标记的情况下鉴定交联将导致交联产物的模糊分配。 13 C标记的光反应性氨基酸的使用被认为优于氘代交联剂的使用，因为可以排除反相色谱中的保留时间偏移。特征碎片离子的观察还增加了交联产物分配的可靠性。 Bpa 对不同氨基酸具有广泛的反应性，并且衍生的距离信息允许映射空间上接近的氨基酸，从而与较长的氘化胺反应性交联剂相比，提供更可靠的蛋白质和蛋白质复合物结构信息，后者通常用于蛋白质 3D 结构分析和蛋白质-蛋白质相互作用研究。
We describe the use of the13C-labeled artificial amino acidp-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein–peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein–protein interaction studies.