LERLIC-MS / MS进行深入表征和谷氨酰胺的定量和天冬酰胺脱酰胺在鸟枪蛋白质组学
Summary
这里，我们提出的长条静电排斥亲水相互作用色谱 – 串联质谱（LERLIC-MS / MS）的方法的一个步骤一步协议。这是一种新的方法，使通过鸟枪蛋白质组学的谷氨酰胺和天冬酰胺脱酰胺亚型的第一次定量和定性。
Abstract
蛋白质脱酰胺化的表征是势在必行破译在人体病理学和其他生化上下文该蛋白的翻译后修饰（PTM）的作用（S）和潜力。为了进行蛋白质脱酰胺的表征，我们最近开发了一种新型长条静电排斥亲水性相互作用色谱 – 串联质谱（LERLIC-MS / MS）方法，其可以分离谷氨酰胺（Gln）和天冬酰胺（ASN）同种型脱酰胺的产物从模型化合物到高度复杂的生物样品。 LERLIC-MS / MS，因此，第一鸟枪蛋白质组学策略的Gln脱酰胺同种型的分离和定量。我们还证明，作为新颖性，在这里列出的样品处理方案稳定琥珀酰亚胺中间体允许由LERLIC-MS / MS表征。如图所示这个视频文章中LERLIC-MS / MS的应用可以帮助阐明目前不明蛋白质脱酰胺的n个分子阵列。此外，LERLIC-MS / MS提供了涵盖不同的生物学背景脱酰胺酶反应的进一步理解。
Introduction
脱酰胺是一种蛋白质翻译后修饰（PTM），通过天冬酰胺（ASN）和/或谷氨酰胺（Gln）残基1的变形例引入了一个负电荷的蛋白质骨架。 1比2：此修改而影响的Asn残基处的共同3生成异构产物异天冬氨酸（异Asp）和正 -天冬氨酸（ASP）。尽管如此，该比率可以通过修复酶L-isoaspartyl甲基（PIMT）3，4的干预而改变。类似地，谷氨酰胺残基的脱酰胺化，生成以预期1异构γ-谷氨酸（γ-Glu），并且的α-谷氨酸亚型（α-GLU）：7的比例3,5，但此比例可通过的作用而移普遍存在的酶的转谷氨酰胺酶2和其它转谷氨酰胺酶，转谷氨酰胺酶包括1，一个电子nzyme最近被确定为与大脑中的6外囊泡。
脱酰胺的起源可以是自发的或酶促的，前者是特别常见的谷氨酰胺残基，其中的转谷氨酰胺酶和其它酶通过转酰氨调解帧间/分子内交联（参见第3对谷氨酰胺酰氨进一步细节及其含义在几种慢性和致命人类疾病）。因此，脱酰胺是具有在所述结构上的一个关键反响和功能的影响分子4，7，8，并且需要进行了深入的化学表征3中的其不同的生物化学后果的光包括其服务老化9的作为分子时钟PTM 。
虽然天冬酰胺残留的脱酰胺已相对充分表征通过自下而上鸟枪蛋白质组学1,10，谷氨酰胺残基的脱酰胺化仍然没有超出由基于电子的自由基碎片11模型化合物的具有挑战性的分析的合适表征方法。我们最近开发了一种新型一维鸟枪蛋白质组学策略（LERLIC-MS / MS）3，使在一次分析中从复杂生物样品和模型化合物谷氨酰胺和天冬酰胺脱酰胺同种型的分离。 LERLIC-MS / MS是基于使用的长长度（50厘米）离子交换柱（LAX）上静电排斥亲水性相互作用色谱（ERLIC）模式下工作的胰蛋白酶消化的肽的分离和耦合到串联质谱（LC- MS / MS）。这个新的分析策略已被用于表征和比较定量每个脱酰胺化残基在人脑组织中的程度F“> 3。然而，这里概述的协议将提供旨在研究蛋白质脱酰胺的特殊性在感兴趣的生化上下文LERLIC-MS / MS的视频成像。
道德守则
本协议的所有程序已通过在新加坡南洋理工大学的机构审查委员会，并根据该机构准则已执行。
Protocol
1.包装长的长度阴离子交换（LAX）毛细管柱 （注意：虽然LAX列可以在家里装，因为我们在这个协议描述，LAX列也可购得，见材料与试剂的表的其他细节）。 暂停50毫克弱阴离子交换包装材料的在3.5mL的包装缓冲器 （ 表1）以制备淤浆。 组装该毛细管柱（50厘米长 – 200微米内径（ID）管）的端部使用一个母对阴配合，一个套圈和阴?…
Representative Results
谷氨酰胺和天冬酰胺残基的脱酰胺化被认为在几种慢性和致命疾病14牵连退行性蛋白修饰（DPM）。已经证明，这种PTM可以预测的抗体和其他分子的半衰期和降解状态在人体内和相似的生物学背景1，15。蛋白质脱酰胺的意义，其实超越了生物医学的背景下，因此这种修改存在于不同的蛋白质组<sup…
Discussion
在该视频文章，我们提出LERLIC-MS / MS 3的步骤一步协议，一种方法来进行深入的表征和准确地确定蛋白质脱酰胺和涉及在此蛋白质修饰的酶过程的程度。 LERLIC-MS / MS是基于静电排斥亲水性相互作用色谱（ERLIC）27的原则下使用的长长度（50厘米）的LAX。使用长长度列，如在我们的研究3中所示，根据它们的等电点27 ERLIC的电位最大…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
，新加坡（-OF-NMRC IRG-0003-2016），和NTU-NHG老化研究资助的国家医学研究理事会（：这项工作是在部分地由教育的新加坡教育部（授予ARC9 / 15级2）资助项目格兰特ARG / 14017）。我们想表达我们的感谢和最诚挚的感谢安德鲁·阿尔珀特博士和PolyLC团队亲切与成为可能这项研究包装材料提供给我们。
Materials
| PolyCAT 3µm 100-Å (bulk material) | PolyLC Inc. | Special order | |
| Long-length ion exchange capillary column 50 cm – 200 µm ID | PolyLC Inc. | Special order | |
| PEEKsil Tubing 1/16" OD x 200 µm ID x 50 cm length | SGE Analytical Science under Trajan Scientific Australia | 620050 | |
| Female-to-female fitting for 1/16" OD tubbing | Upchurch Scientific | UPCHF-125 | |
| Female nut for microferule | Upchurch Scientific | UPCHP-416 | |
| Microferule | Upchurch Scientific | UPCHF-132 | |
| Pressure Bomb NanoBaume | Western Fluids Engineering | SP-400 | |
| Shimadzu Prominence UFLC system | Shimadzu | Prominence UFLC | |
| Bullet Blender | Next Advance | BBX24 | |
| Safe-lock tubes | Eppendorf | T9661-1000EA | |
| Stainless steel beads. 0.9 – 2.0 mm. 1 lb. Non-sterile. | Next Advance | SSB14B | |
| Table-top centrifuge | Hettich Zentrifugen | Rotina 380 R | |
| Standard Digital Heated Circulating Bath, 120VAC | PolyScience 8006 6L | 8006A11B | |
| Sep-pack c18 desalting cartridge 50 mg | Waters | WAT020805 | |
| Vacumm concentrator | Eppendorf | Concentrator Plus System | |
| Dionex UltiMate 3000 UHPLC | Dionex | UltiMate 3000 UHPLC | |
| Orbitrap Elite mass spectrometer | Thermo Fisher Scientific Inc. | ORBITRAP ELITE | |
| Michrom Thermo CaptiveSpray | Michrom-Bruker Inc. | TCSI-SS2 | |
| Incubator INCUCELL | MMM Group | INCUCELL111 | |
| Sequencing-grade modified trypsin | Promega | V5111 | |
| Protease inhibitor cocktail tablets | Roche | 11836170001 (ROCHE) | |
| Phosphate buffer solution 10X (diluted to 1x) | Sigma-Aldrich | P5493 | |
| Ammonium acetate | Sigma-Aldrich | A1542 | |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | |
| Dithiothreitol | Sigma-Aldrich | D0632 | |
| Iodoacetamide | Sigma-Aldrich | i6125 | |
| Formic acid | Sigma-Aldrich | F0507 (HONEYWELL) | |
| Ammonium hydroxide | Sigma-Aldrich | 338818 (HONEYWELL) | |
| Acetonitrile HPLC grade | Sigma-Aldrich | 675415 | |
| Isopropanol HPLC grade | Sigma-Aldrich | 675431 | |
| Water HPLC grade | Sigma-Aldrich | 14263 |
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Citazione di questo articolo
Gallart-Palau, X., Serra, A., Sze, S. K. LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics. J. Vis. Exp. (122), e55626, doi:10.3791/55626 (2017).