Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry.
Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry.
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DOI：
10.1021/ac900853k
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发表时间：
2009-07-01
影响因子：
7.4
通讯作者：
Adkins, Joshua N.
中科院分区：
文献类型：
--
作者：
Chowdhury, Saiful M.;Du, Xiuxia;Tolic, Nikola;Wu, Si;Moore, Ronald J.;Mayer, M. Uljana;Smith, Richard D.;Adkins, Joshua N.
化学交联与质谱学相结合是鉴定蛋白质-蛋白质相互作用和限制蛋白质结构的有效方法。然而，在进行质谱分析之前，交联肽的富集化对于降低样品的复杂性至关重要。此外，紧凑型交联剂通常用于提供较短的间隔基长度、蛋白质复合体的表面可及性，并且在天然络合物结构稳定的条件下必须具有合理的溶解性。在这项研究中，我们提出了一种新型的紧凑交联剂，它包含两个不同的特征：1)乙炔标记和2)小分子检测标记(NO2-)，以保持在水中的合理溶解度。在使用炔-叠氮点击化学偶联亲和标签之后，炔标签能够在蛋白水解性切割之后使交联肽得以丰富。小NO2-部分的中性损失为MS/MS分析中检测交联肽提供了二次手段，增加了对多肽鉴定的信心。我们展示了这种交联剂的标记效率，我们使用泛素将其命名为CLIP(Click-Enabled Linker for Interaction Proteins)。CLIP对高度复杂的大肠杆菌细胞裂解物中的交联型泛素具有丰富的能力。还展示了用于改进交联肽的自动识别的相互交联肽(由交联剂连接的两个肽)的顺序CID-MS/MS和ETD-MS/MS。
Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides.
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影响因子：
4.4
作者：
Anderson, Gordon A.;Tolic, Nikola;Bruce, James E.
通讯作者：
Bruce, James E.
影响因子：
5.6
作者：
Haririnia, Aydin;D'Onofrio, Mariapina;Fushman, David
通讯作者：
Fushman, David
DOI：
10.1073/pnas.0607084104
发表时间：
2007-02-13
影响因子：
11.1
作者：
Chi, An;Huttenhower, Curtis;Hunt, Donald F.
通讯作者：
Hunt, Donald F.
影响因子：
7.4
作者：
YATES, JR;ENG, JK;SCHIELTZ, D
通讯作者：
SCHIELTZ, D
影响因子：
48
作者：
Rinner, Oliver;Seebacher, Jan;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi