使用膜内消化技术评估蛋白质与蛋白质相互作用
Summary
在这里,我们提出了一个方案,用于制备质谱样本的膜上消化技术。该技术有助于方便分析蛋白质与蛋白质的相互作用。
Abstract
许多细胞内蛋白质根据细胞内和细胞外情况进行物理交互。事实上,细胞功能很大程度上依赖于细胞内蛋白质与蛋白质的相互作用。因此,关于这些相互作用的研究对于促进对生理过程的理解是必不可少的。相关蛋白质的共沉淀,然后进行质谱分析(MS),能够识别新的蛋白质相互作用。在这项研究中,我们详细介绍了免疫沉淀-液相色谱(LC)-MS/MS分析与膜内消化相结合,用于蛋白质与蛋白质相互作用的分析。该技术适用于粗免疫沉淀物,可提高蛋白组分析的通量。标记重组蛋白是使用特定抗体沉淀的;其次,在聚氯丁二烯膜片片上被斑点的免疫沉淀物进行还原烷基化。在胰蛋白酶化后,使用LC-MS/MS对消化的蛋白质残留物进行分析。因此,该方法对新型蛋白质与蛋白质相互作用的表征是方便和有用的。
Introduction
虽然蛋白质在生物体中起着构成作用,但它们在细胞内环境中不断被合成、加工和降解。此外,细胞内蛋白质经常在物理和生物化学上相互作用,这会影响一个或两个1,2,3的功能。例如,将与plissome相关的蛋白同源性CWC22与真核转化启动因子4A3(eIF4A3)直接结合是组装外子结复合物4所必需的。与此一致,一种对CWC22缺乏亲和力的eIF4A3突变体未能促进外子结复合驱动的mRNA拼接4。因此,蛋白质相互作用的研究对于准确理解生理调节和细胞功能至关重要。
质谱学(MS)的最新进展已应用于蛋白质-蛋白质相互作用的综合分析。例如,内源性蛋白质或外源引入标记蛋白及其相关蛋白的共沉淀,然后进行MS分析,能够识别新的蛋白质相互作用5。然而,MS/MS分析的一个主要瓶颈是蛋白质样品的胰蛋白消化不良恢复。用于对细胞解物进行蛋白体分析,通常采用凝胶内和膜内消化技术来制备MS/MS样品。我们之前将凝胶内消化程序与膜内消化技术6进行了比较,并表明后者与更好的序列覆盖率有关。聚苯乙烯二氟化酶 (PVDF) 膜可能适合于此目的,因为它机械坚固,耐高浓度有机溶剂7、8,允许固定的酶消化蛋白质在存在80%醋酸酯9。此外,膜上的固定可以诱导目标蛋白的构象变化,导致胰体消化效率的提高10。因此,在本文中,我们描述了使用膜内消化技术对蛋白质相互作用进行免疫沉淀-LC/MS/MS分析的使用。这种简单的方法便于分析蛋白质-蛋白质相互作用,即使在非专业实验室。
Protocol
Representative Results
Discussion
我们之前已经描述了使用LC-MS/MS在膜上消化技术6之前,在氧化的低密度脂蛋白中对脂蛋白B-100的氧化修饰的分析。在本研究中,我们将该技术与免疫沉淀相结合,并确定了几种与钙痛-6相关的蛋白质。这种新技术代表了筛选候选相关蛋白质的便捷方法。Calpain-6是钙蛋白酶家族11的非蛋白解体成员,据报道,它通过其蛋白质-蛋白质相互作用12、13<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究部分得到了日本科学促进协会KaKENHI赠款号17K09869(至AM)、日本科学促进会KaKENHI赠款号15K09418(至TM)的支持,这是来自Kanehara Ichiro医学科学基金会的一笔研究补助金。和苏津纪念基金会的研究补助金(全部给TM)。
Materials
| Acetonitrile | Wako | 014-00386 | |
| Citric acid | Wako | 030-05525 | |
| DiNA | KYA Tech Co. | nanoflow high-performance liquid chromatography | |
| DiNa AI | KYA Tech Co. | nanoflow high-performance liquid chromatography equipped with autosampler | |
| DTT | Nacalai tesque | 14112-94 | |
| Dynabeads protein G | Thermo Fisher Scientific | 10003D | |
| Formic acid | Wako | 066-00461 | |
| HiQ Sil C18W-3 | KYA Tech Co. | E03-100-100 | 0.10mmID * 100mmL |
| Iodoacetamide | Wako | 095-02151 | |
| Lipofectamine 3000 | Thermo Fisher Scientific | L3000008 | |
| Living Colors A.v. Monoclonal Antibody (JL-8) | Clontech | 632380 | |
| NaCl | Wako | 191-01665 | |
| NH4HCO3 | Wako | 018-21742 | |
| Nonidet P-40 | Sigma | N6507 | poly(oxyethelene) octylphenyl ether (n=9) |
| peptide standard | KYA Tech Co. | tBSA-04 | tryptic digests of bovine serum albumin |
| PP vial | KYA Tech Co. | 03100S | plastic sample tube |
| Protease inhibitor cooctail | Sigma | P8465 | |
| ProteinPilot software | Sciex | 5034057 | software for protein identification |
| Sequencing Grade Modified Trypsin | Promega | V5111 | trypsin |
| Sodium orthovanadate | Sigma | S6508 | |
| Sodium phosphate dibasic dihydrate | Sigma | 71643 | |
| TFA | Wako | 206-10731 | |
| trap column | KYA Tech Co. | A03-05-001 | 0.5mmID * 1mmL |
| TripleTOF 5600 system | Sciex | 4466015 | Hybrid quadrupole time-of-flight tandem mass spectrometer |
| Tris | Wako | 207-06275 | |
| Tween-20 | Wako | 160-21211 |
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