Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.
Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.
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DOI：
10.1371/journal.pone.0242376
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发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Wang MM
中科院分区：
文献类型：
--
作者：
Cartee NMP;Lee SJ;Keep SG;Wang MM
蛋白质巯基残基参与关键的结构和生物化学功能。由可逆氧化还原反应或永久共价封端调节的巯基状态的改变可能难以识别。为了推进蛋白质巯基的检测，我们描述了新的兔单克隆抗体的生产，与氨基甲酰基-半胱氨酸（CAM-cys），碘乙酰胺（IAM）标记的蛋白质巯基残基的产物反应。这些抗体与用IAM标记的蛋白质结合（但不与N-乙基马来酰亚胺（NEM）或丙烯酰胺结合），并且当应用于用IAM处理的细胞裂解物的蛋白质印迹时鉴定多个蛋白质条带。单克隆抗体标记CAM-cys修饰的肽序列和纯化的蛋白质（人血管性血友病因子（基因：vWF）、Jagged 1（基因：JAG 1）、层粘连蛋白亚基α 2（基因：LAMA 2）、血小板反应蛋白-2（基因：TSP 2）和胶原蛋白IV（基因：COL 4））的子集，但不识别特定蛋白质，如牛血清白蛋白（基因：BSA）和人血小板反应蛋白-1（基因：TSP 1）、双糖蛋白聚糖（基因：BGN）和核心蛋白聚糖（基因：DCN）。用于产生CAM-cys抗体的肽序列的扫描突变体阐明了背景依赖性反应性所需的残基。除了识别体外标记的蛋白质外，抗体还用于鉴定来自用IAM脉冲标记的活细胞的选定的含巯基蛋白质。新的CAM-cys单克隆抗体与其他生物化学工具结合的进一步发展可以补充目前的方法，用于特定蛋白质内的巯基检测。此外，当需要标记蛋白质亚群时，CAM-cys反应性试剂可能是有用的。
Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.
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影响因子：
2.9
作者：
Zhang, Xiaojie;Lee, Soo Jung;Young, Kelly Z.;Josephson, David A.;Geschwind, Michael D.;Wang, Michael M.
通讯作者：
Wang, Michael M.
影响因子：
1.2
作者：
Hoch, Dominic G.;Abegg, Daniel;Adibekian, Alexander
通讯作者：
Adibekian, Alexander
影响因子：
4
作者：
Wang, Ke;Peng, Hanjing;Wang, Binghe
通讯作者：
Wang, Binghe
影响因子：
7.4
作者：
Hill, Bradford G.;Reily, Colin;Oh, Joo-Yeun;Johnson, Michelle S.;Landar, Aimee
通讯作者：
Landar, Aimee
影响因子：
10.4
作者：
Hogg, P. J.
通讯作者：
Hogg, P. J.