Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.
Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.
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DOI：
10.1002/rcm.4066
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发表时间：
2009-06
影响因子：
2
通讯作者：
Prevelige, Peter E., Jr.
中科院分区：
文献类型：
--
作者：
Kang, Sebyung;Mou, Liyuan;Lanman, Jason;Velu, Sadanandan;Brouillette, Wayne J.;Prevelige, Peter E., Jr.
化学交联结合质谱已被用来阐明蛋白质结构和蛋白质-蛋白质相互作用。然而，样品的异质性和交联肽的相对低丰度使得这种方法具有挑战性。为了克服这一障碍，我们合成了赖氨酸反应性同双功能交联剂，其中生物素在接头中间，并将其用于富集交联肽。生物素标记的交联剂与纯化的HIV-1 CA的反应导致形成悬挂和分子内交联。使用链霉亲和素包被的板和质谱友好缓冲液有效地富集和回收用生物素化交联剂修饰的肽。修饰肽的富集和主要未修饰肽的去除简化了质谱及其分析。FT-ICR MS的高质量准确度和线性离子阱的MS/MS能力相结合，使我们能够明确识别交联位点和其他修饰，如氧化。
Chemical cross-linking combined with mass spectrometry has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them for enriching cross-linked peptides. The reaction of biotin tagged cross-linkers with purified HIV-1 CA resulted in the formation of hanging and intra-molecular cross-links. The peptides modified with biotinylated cross-linkers were effectively enriched and recovered using a streptavidin coated plate and mass spectrometry friendly buffers. The enrichment of modified peptides and removal of the dominantly unmodified peptides simplify mass spectra and their analyses. The combination of high mass accuracy of FT-ICR MS and MS/MS capability of linear ion trap allows us to unambiguously identify the cross-linking sites and additional modification, such as oxidation.
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DOI：
10.1073/pnas.240326597
发表时间：
2000-12-19
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
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