Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents

A major
challenge in cross-linking/mass spectrometry (MS) is targeting
carboxyl functions in proteins under physiological conditions that
do not disturb the protein’s conformation. Cross-linking of
glutamic acid and aspartic acid residues in proteins will greatly
expand the scope of structural mass spectrometry. We discovered that
carboxyl-reactive cross-linkers have already been employed for many
years in cross-linking/MS studies, yet in a completely different context.
Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine
cross-linkers, are currently considered to react nonspecifically upon
UV-A photoactivation with all 20 proteinogenic amino acids through
a reactive carbene that inserts mainly into C–H bonds. We discovered
that the cross-linking capability of diazirines based on X–H
(X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines
isomerize to linear diazo compounds that can react with carboxylic
acids to yield esters. On top of that, the resulting cross-linked
products are MS-cleavable allowing an automated analysis of cross-links
via customized software tools. Therefore, diazirines open an entirely
new route for photo-cross-linking of carboxylic acids. Previous cross-linking
studies using diazirines have to be revisited in the light of these
findings.

交联/质谱（cross-linking/mass spectrometry, MS）领域的核心挑战之一，是在不干扰蛋白质构象的生理条件下，靶向蛋白质中的羧基官能团。对蛋白质内谷氨酸与天冬氨酸残基进行交联反应，将极大拓展结构质谱学的研究范围。我们发现，羧基反应性交联剂其实已在交联/MS研究中应用多年，却始终处于完全不同的应用语境中。
当前学界普遍认为，基于双吖丙啶（diazirine）的交联剂——例如光蛋氨酸（photomethionine）与琥珀酰亚胺基双吖丙啶（succinimidyldiazirine）交联剂——在紫外A（UV-A）光活化后，会通过主要插入碳氢键（C–H bonds）的活性卡宾，与全部20种蛋白质源性氨基酸（proteinogenic amino acids）发生非特异性反应。我们的研究表明，双吖丙啶通过X-H（X=C、N、O）插入实现的交联能力，实则仅为冰山一角。双吖丙啶可异构化为线型重氮化合物，后者能够与羧酸反应生成酯类产物。
尤为关键的是，所得交联产物具备质谱可裂解（MS-cleavable）特性，可通过定制化软件工具实现交联位点的自动化分析。因此，双吖丙啶为羧酸类物质的光交联开辟了一条全新路径。基于上述发现，过往所有使用双吖丙啶的交联研究均需重新审视。