使用阿基-树脂辅助捕获检测蛋白质S-囊肿
Summary
Acyl-RAC（阿西尔-树脂辅助捕获）是一种高度敏感、可靠且易于执行的方法，用于检测各种生物样品中半胱氨酸残留物（S-acycyy）的可逆脂改性。
Abstract
蛋白质S-囊化，也称为S-Palmiyyyy化，是通过小链脂肪酸，通过小链脂肪酸对半胱氨酸残留物进行可逆的转化后修饰。S-acy化正在成为一种广泛的调节机制，它可以调节蛋白质生物活性的所有方面，从复杂的形成到蛋白质的贩运和蛋白质的稳定性。最近对蛋白质S-脱化生物功能的理解取得了进展，这主要是因为新型生化工具的开发，使各种生物样品中蛋白质S-脱化能够进行可靠和灵敏的检测。在这里，我们描述了乙基树脂辅助捕获（Acyl-RAC），这是一种最近开发的方法，基于通过硫醇反应性Sepharose珠选择性捕获内质S-囊肿蛋白。与现有方法相比，Acyl-RAC 需要较少的步骤，当与质谱法相结合以识别新型 S-循环目标时，可以产生更可靠的结果。该技术的一个主要限制是缺乏通过同一硫酯键区分附着在半胱氨酸上的脂肪酸物种的能力。
Introduction
S-脱硫是一种可逆的翻译后修饰，涉及通过拉迪尔硫酯键1在靶蛋白上加入一个脂肪酸酶链到目标蛋白上的内半胱氨酸残留物。它最初被报道为用棕榈酸，一种饱和脂肪酸2的蛋白质的修饰，因此这种修饰通常被称为S-棕榈酸。除了棕榈酸盐，蛋白质可以通过各种更长和较短的饱和（硫酸和凝结酸）、单不饱和（油酸盐）和多不饱和（烷基酮和异氨酯酸）脂肪酸33、4、5、6、74,5,6,7可逆性地改变。在真核细胞中，S-acycye是由一种称为DHHC蛋白酶的酶家族催化的，半胱氨酸脱硫酶的反向反应被蛋白硫酯酶催化，其中大多数仍然保持神秘的8。
硫化物键的可浸性使这种脂质修饰可逆，使其能够动态调节蛋白质簇簇、血浆膜定位、细胞内贩运、蛋白质-蛋白质相互作用和蛋白质稳定性9,9、10。因此，S-囊肿与亨廷顿舞蹈症、阿尔茨海默病和几种癌症（前列腺、胃癌、膀胱癌、肺癌、结肠直肠癌）等疾病有关，因此需要开发可靠的方法来检测这种转化后蛋白修饰11。
代谢标记与放射性（[3H]，[14C]或[125I]）棕榈酸是最早开发的方法之一，以测定蛋白质S-囊14化12，13，14。12,13,14然而，基于放射性标签的方法存在健康问题，不是很敏感，耗时，只检测高丰富的蛋白质15的脂质化。一种更快、非放射性的放射性替代无线电标签是代谢标签与生物正交脂肪酸探针，这是例行用于检测蛋白质S-囊肿16的动态。在这种方法中，含有化学报告器（烷基或阿齐德组）的脂肪酸通过蛋白质转质酶被结合到S-青化蛋白中。Azide-alkyne Huisgen环增反应（点击化学）然后可用于将一个功能化组，如氟磷或生物素，连接到一体化脂肪酸，允许检测S-囊青蛋白17，18，19。17,18,19
Acyl-生物锡交换（ABE）是广泛采用的生物化学方法之一，用于捕获和鉴定S-囊肿蛋白，绕过代谢标记的一些缺点，如不适合组织样本15。该方法可用于分析各种生物样品的S-化，包括组织和冷冻细胞样本20、21。20,该方法基于中性羟基胺对酸基组和半胱氨酸残留物之间的三酯键的选择性裂解。然后，用三醇反应生物组子捕获。生成的生物微化蛋白然后使用链球菌加罗丝进行亲和力纯化，并通过免疫凝血进行分析。
一种称为乙基树脂辅助捕获（Acyl-RAC）的替代方法后来被引入，用游脱反应树脂22，23,23取代生物微化步骤，直接结合游自由半胱氨酸。与ABE相比，这种方法的步骤较少，同样可用于检测各种样品1的蛋白质S-青化。
Acyl-RAC 由 4 个主要步骤（图 1） 组成 。
1. 阻止自由三醇组;
2. 选择性地裂解半胱氨酸-酸酯结合与中性羟基胺（HAM），以暴露半胱氨酸硫醇群;
3. 用三醇反应性树脂捕获脂质化半胱氨酸;
4. 洗脱后选择性富集S-青化蛋白，减少缓冲。
然后，通过免疫凝血或进行基于质谱（MS）的质谱（MS）分析捕获的蛋白质，以评估不同种类和组织22、24、25的S-cycytttome。22,24,25通过对捕获的蛋白质的胰蛋白消化和LC-MS/MS22分析产生的肽，也可以识别个别S-青化位点。在这里，我们演示了如何使用 acyl-RAC 同时检测细胞系和组织样本中多个蛋白质的 S-acy 化。
Protocol
Representative Results
Discussion
在这里，我们成功地利用acyl-RAC测定检测培养的人类细胞和从小鼠组织中提取的原发细胞中选定蛋白质的S-化。该方法简单、灵敏，使用标准生物化学技术，在设备要求极小的情况下，易于执行。该方法已被证明能够成功地识别新的S-囊肿蛋白，如蛋白质转移系统（Sec61b）、核糖蛋白S11（Rps11）和微体谷胱甘肽-S-转移酶3（MGST3）22。22acyl-RAC的敏感性和适应性使其适合在各种?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院提供的5R01GM115446和1R01GM130840的支持。
Materials
| cOmplete Protease Inhibitor Cocktail tablets | Sigma | 11836170001 | |
| Eppendorf Centrifuge 5424 | Eppendorf | 22620444 | |
| Hydroxylamine (HAM) | Sigma | 159417 | |
| Methyl methanethiosulfonate (MMTS) | Sigma | 64306 | |
| Mini tube rotator | LabForce | ||
| ML211 | Cayman | 17630 | |
| Multi-Therm Cool-Heat-Shake | Benchmark Scientific | H5000-HC | |
| n-Dodecyl β-D-maltoside (DDM) | Sigma | D641 | |
| Phosphatase Inhibitor Cocktail 2 | Sigma | P5726 | |
| Thiopropyl-Sepharose 6B (TS) | Sigma | T8387 | |
| Ultrasonics Quantrex Sonicator | L & R |
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