Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes.
Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes.
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DOI：
10.1021/ac9017132
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发表时间：
2009-11-01
影响因子：
7.4
通讯作者：
Zhou, Zhaohui Sunny
中科院分区：
文献类型：
--
作者：
Zang, Tianzhu;Dai, Shujia;Chen, Dajun;Lee, Bobby W. K.;Liu, Suli;Karger, Barry L.;Zhou, Zhaohui Sunny
血液中同型半胱氨酸(Hcy)水平升高、高同型半胱氨酸血症或同型半胱氨酸尿症与各种疾病和条件有关。同型半胱氨酸硫内酯(Hcy TL)是同型半胱氨酸的代谢物，与蛋白质中的胺基反应形成稳定的酰胺类、同型半胱氨酸酰胺或N-同型半胱氨酸化的蛋白质。已有研究表明，蛋白N-同型半胱氨酸化参与了Hcy升高的细胞毒性作用。由于其异质性和相对较低的丰度，检测这种翻译后修饰仍然具有挑战性。另一方面，同型半胱胺中的伽马-氨基硫醇基团具有不同于天然蛋白质的化学反应活性。在中等酸性条件下，氨基硫醇不可逆地与醛按化学计量反应生成稳定的1，3-噻嗪，而天然蛋白质中醛和氨基之间的可逆希夫碱的形成由于胺的质子化而明显受阻。因此，我们开发了高度选择性的化学方法来衍生化带有各种醛标记的N-同型半胱氨酸化蛋白质，从而便于后续的分析。例如，结合凝胶电泳的荧光或生物素标记允许对复杂的生物样本进行量化和全球图谱分析，例如来自大鼠、小鼠和人类的血红蛋白和血浆；用醛树脂进行亲和浓缩大大降低了样品的复杂性。此外，还观察到血红蛋白中赖氨酸残基对Hcy TL的不同反应性。
Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this post-translational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin towards Hcy TL were observed.
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影响因子：
2.9
作者：
Perla-Kajan, Joanna;Marczak, Lukasz;Jakubowski, Hieronim
通讯作者：
Jakubowski, Hieronim
影响因子：
5.7
作者：
Finkelstein, JD
通讯作者：
Finkelstein, JD
DOI：
10.1073/pnas.191223098
发表时间：
2001-09-25
影响因子：
11.1
作者：
Hilgers, MT;Ludwig, ML
通讯作者：
Ludwig, ML
影响因子：
4.8
作者：
Jakubowski, H
通讯作者：
Jakubowski, H
影响因子：
158.5
作者：
De Wals, Philippe;Tairou, Fassiatou;Niyonsenga, Theophile
通讯作者：
Niyonsenga, Theophile