Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.
Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.
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DOI：
10.2144/000114516
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发表时间：
2017-02-01
期刊：
影响因子：
2.7
通讯作者：
Hemsley PA
中科院分区：
文献类型：
--
作者：
Hurst CH;Turnbull D;Plain F;Fuller W;Hemsley PA
S-棕榈酰化（S-酰化）是一种新出现的蛋白质半胱氨酸残基的动态翻译后修饰。目前用于蛋白S-棕榈酰化的测定涉及S-棕榈酰基的体内标记或化学裂解，以揭示随后可以用亲和手柄（酰基交换）标记的游离半胱氨酸巯基。因此，使用酰基交换化学进行的蛋白质S-棕榈酰化的测定需要阻断非S-棕榈酰化的半胱氨酸，通常使用N-乙基马来酰亚胺，以防止非特异性检测。这又需要多个基于沉淀的净化步骤来去除阶段之间的试剂，通常导致可变的样品损失、信号降低或蛋白质聚集。这些联合收割机降低了这些测定的灵敏度、可靠性和准确性，并且还需要大量的时间来执行。通过在水性Diels-Alder 4+2环加成反应中用2，3-二甲基-1，3-丁二烯对N-乙基马来酰亚胺的化学清除取代这些沉淀步骤，可以大大提高灵敏度和准确度，同时减少测定所需的动手时间和总时间。
S-palmitoylation (S-acylation) is an emerging dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labelling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labelled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide, to prevent non-specific detection. This in turn necessitates multiple precipitation based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal or protein aggregation. These combine to reduce the sensitivity, reliability and accuracy of these assays and also requires a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of N-ethylmaleimide by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction it is possible to greatly improve sensitivity and accuracy while reducing hands-on and overall time required for assays.