Hydrazide reactive peptide tags for site-specific protein labeling.
Hydrazide reactive peptide tags for site-specific protein labeling.
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DOI：
10.1021/bc200415v
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发表时间：
2011-10-19
影响因子：
4.7
通讯作者：
Weiss, Gregory A.
中科院分区：
文献类型：
--
作者：
Eldridge, Glenn M.;Weiss, Gregory A.
针对特定短肽的新的位点特异性蛋白质标记(SSPL)反应可用于实时检测活细胞内的蛋白质。一种SSPL方法将生物正交试剂与互补多肽相匹配。在这里，使用基于反应的选择，从噬菌体展示文库中选择酰肼反应性多肽。选择条件包括不同pH的洗涤和NaCNBH3处理，以便专门选择含有活性羰基的多肽。将选定的多肽与T4溶菌酶融合或在滤纸上合成，用于比色分析多肽与肼的相互作用。多肽-溶菌酶蛋白融合展示了粗制细菌细胞裂解物中的酰肼反应(HYRE)多肽的特定共价标记，足以特异性地检测过度表达的蛋白融合。化学合成一个短的Hyre标签变异体，随后与两个结构不同的肼探针反应生成共价加合物，通过MALDI-TOF MS和MS/MS观察到，我们没有分离出含有反应性羰基的多肽，而是观察到了与Hyre标签114的N末端组氨酸的反应，该序列的氨基酸序列HKSNHSSKNRE在中性pH下攻击肼的羰基。然而，在选择洗涤步骤中使用的pH值(<；6.0)时，会形成另一种含亚胺的产品，该产品可以用氰化硼氢化钠还原。MSMS进一步揭示，这种低pH产物在Ser6上形成加合物。本文所述的新型双分子反应的进一步优化可以为体内蛋白质标记和生物偶联化合物的合成提供有用的工具。所报道的筛选方法可广泛应用于用生物正交试剂进行其他位点特异性蛋白质标记反应的多肽的鉴定。
New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents.
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影响因子：
15
作者：
Halo TL;Appelbaum J;Hobert EM;Balkin DM;Schepartz A
通讯作者：
Schepartz A
影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
影响因子：
56.9
作者：
CHALFIE, M;TU, Y;PRASHER, DC
通讯作者：
PRASHER, DC
影响因子：
14.8
作者：
Hilpert, Kai;Winkler, Dirk F. H.;Hancock, Robert E. W.
通讯作者：
Hancock, Robert E. W.
DOI：
10.1073/pnas.0611713104
发表时间：
2007-03-06
影响因子：
11.1
作者：
Hauser, Christina T.;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.