利用毛细管区电泳串联质谱技术进行大尺度自上而下蛋白质组学
Summary
通过毛细管区电泳-电喷雾-串联质谱 (CZE-proteoforms), 描述了蛋白质样品中的分离、鉴定和表征的详细协议。该协议可用于简单蛋白样品中 proteoforms 的高分辨率表征和复杂蛋白质组样本中 proteoforms 的大尺度鉴定。
Abstract
毛细管区电泳-电喷雾电离-串联质谱 (CZE) 已被公认为自上而下蛋白质组学的有用工具, 旨在表征复杂蛋白质组中的 proteoforms。然而, CZE 在大范围自上而下蛋白质组学中的应用由于 CZE 的样品加载能力低, 分离窗口狭窄而受阻。在这里, 使用 CZE 的协议描述了一个微升的样品加载量和一个90分钟的分离窗口, 用于大规模自上而下的蛋白质组学。CZE 平台基于线性聚丙烯酰胺 (LPA) 涂层分离毛细管, 具有极低的电渗流, 一种基于动态 pH 结的在线样品浓缩方法, 具有高效率的蛋白质堆积,电动力学泵鞘流 CE 接口具有极高的灵敏度, 以及具有高质量分辨率和扫描速度的离子阱质谱仪。该平台可用于简单完整蛋白样品的高分辨率表征和各种复杂蛋白质组中 proteoforms 的大尺度表征。例如, 我们演示了一种高效分离标准蛋白混合物和使用该平台对许多杂质进行高度灵敏检测的方法。作为另一个例子, 这个平台可以产生超过 500 proteoform 和190蛋白质鉴定从大肠杆菌蛋白质组在一个单一的 CZE ms/ms 运行。
Introduction
自上而下的蛋白质组学 (TDP) 的目的是大规模表征 proteoforms 内的蛋白质。TDP 依赖于在电喷雾电离-串联质谱 (ESI ms/ms) 分析之前, 由于蛋白质组1、2 的高复杂性和大浓度动态范围而对完整蛋白进行有效液相分离 ,3,4,5。毛细管区电泳 (CZE) 是一种强大的技术, 以分离生物分子的大小-电荷比6。CZE 是相对简单的, 只需要一个开放管状熔融石英毛细管, 背景电解质 () 和电源。完整的蛋白质样品可以通过压力或电压加载到毛细管中, 并通过浸泡毛细管两端并施加高电压来启动分离。CZE 可接近超高分离效率 (> 100万个理论板), 用于分离生物分子7。CZE 的灵敏度比广泛使用的反相液相色谱 (RPLC)-ms 对完整蛋白8的分析有了极大的提高。虽然 CZE 对大规模自上而下蛋白质组学有很大的潜力, 但它在蛋白质组学中的广泛应用受到了一些问题的阻碍, 包括低采样量和窄分离窗口。CZE 中典型的样品加载量约为总毛细管体积的 1%, 通常对应于小于 100 nL9、10、11。由于强电渗流 (EOF)9、10, CZE 的分离窗口通常小于30分钟。这些问题限制了 CZE (ms), 用于识别来自复杂蛋白质组的大量 proteoforms 和低丰 proteoforms。
通过在线样品浓度法 (例如固相微萃取 [固相微萃取]12、13、场增强样品堆积 (CZE)9 , 提高了样品加载量。,11,14、动态 pH 结15、16、17、18)。和动态 ph 结比固相微萃取简单, 只需要在电导率和 pH 值之间有显著差异的样品缓冲液。在取样区域和毛细管中的的小区之间的边界上, 该样品缓冲液的电导率要低得多。动态 pH 结使用一个基本的样品塞 (例如, 50 毫米碳酸氢钠, ph 8) 和酸盐 (例如, 5% [v-/v] 醋酸, pH 2.4) 在样品插头的两侧。在毛细管的注射端施加高正压时, 会发生基本样品塞的滴定, 在进行 CZE 分离之前将分析物集中在紧密的插头上。最近, Sun 集团系统地比较了在完整的蛋白质在线堆叠的承认和动态 ph 结, 表明动态 ph 结可以产生更好的性能比坦白的在线浓度完整的蛋白质时样品注射量为总毛细管体积19的25%。
中立涂层分离毛细血管 (例如, 线性聚丙烯酰胺 [LPA]) 已被用于减少毛细管中的 EOF, 减慢 CZE 分离和扩大分离窗口20,21。最近, Dovichi 集团开发了一个简单的程序, 在毛细血管内壁上制备稳定的 LPA 涂层, 利用过硫酸铵 (ap) 作为引发剂和温度 (50 摄氏度) 自由基生产和聚合22.最近, Sun 集团采用了 LPA 涂层分离毛细管和动态 pH 结方法 CZE 分离完整的蛋白质, 达到微升的样品加载量和90分钟的分离窗口19。这个 CZE 系统打开门使用 CZE ms/ms 的大规模自上而下的蛋白质组学。
CZE 需要一个高度健壮和敏感的接口, 以耦合 CZE 到 MS。三 ce ms 接口在 ce 的历史中得到了很好的开发和商业化, 它们是共轴鞘流接口23, sheathless 接口使用多孔尖端作为 ESI 发射器24, 以及电动力学泵送护套流量接口25,26。电动力学泵鞘-流界面 CZE-ms 已达到低 zeptomole 肽检测限制9, 超过1万肽鉴定 (IDs) 从 HeLa 细胞蛋白质组在单一运行14, 快速表征完整的蛋白质11, 和高度稳定和可重现的生物分子分析26。最近, 采用 LPA 包覆分离毛细管、动态 pH 结法和电动力学泵鞘流界面, 用于大肠埃希氏杆菌 (大肠杆菌) 蛋白质组 19 的大尺度上向下蛋白分析 ,27。CZE 平台通过与尺寸排除色谱 (SEC)-RPLC 分馏27的耦合, 在单个运行19和近 6000 proteoform id 中接近 500 proteoform id。结果清楚地显示了 CZE 对大范围自上而下蛋白质组学的能力。
本文介绍了一种用于大规模自上而下蛋白质组学的 CZE/ms 的详细过程。CZE 系统采用了 LPA 涂层毛细管, 以减少毛细管中的 EOF, 动态 pH 结方法的蛋白质在线浓度, 电动力学泵鞘流接口 CZE 耦合到 MS, orbitrap 质量用于采集蛋白质的 ms 和 ms/ms 光谱的光谱仪, 以及通过数据库搜索 Proteoform ID 的 TopPIC (自上而下的质谱 Proteoform 识别和表征) 软件。
Protocol
Representative Results
Discussion
在这里, 我们提供了一个详细的协议, 使用 CZE 的高分辨率表征 proteoforms 在简单的蛋白质样本和大规模鉴定 proteoforms 在复杂的蛋白质组样品。图 1显示了 CZE-ESI/ms 系统的示意图。协议中有四关键步骤。首先, 在分离毛细管的内壁上制备高质量的 LPA 涂层非常重要。用 LPA 包覆分离毛细管可以减少毛细管中的 EOF, 拓宽 CZE 的分离窗, 减少其内壁19的蛋白质吸附。<em…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢 Heedeok 在密歇根州立大学化学系的小组, 为实验提供大肠杆菌细胞. 作者感谢全国普通医学研究院、国立卫生研究院 (NIH) 通过授予 R01GM118470 (向 x. 刘) 和授予 R01GM125991 (对 l. 孙和 x. 刘) 的支持。
Materials
| Fused silica capillary | Polymicro Technologies | 1068150017 | 50 µm i.d. 360 µm o.d. |
| Sodium hydroxide pellets | Macron Fine Chemicals | 7708-10 | Corrosive |
| LC-MS grade water | Fisher Scientific | W6-1 | |
| Hydrochloric acid | Fisher Scientific | SA48-1 | Corrosive |
| Methanol | Fisher Scientific | A456-4 | Toxic, Health Hazard |
| 3-(Trimethoxysilyl)propyl methacrylate | Sigma-Aldrich | M6514 | Moisture and heat sensitive |
| Hydrofluoric acid | Acros Organics | 423805000 | Highy toxic |
| Acrylamide | Acros Organics | 164855000 | Toxic, health hazard |
| Ammonium persulfate | Sigma-Aldrich | A3678 | Health hazard, Oxidizer |
| lysozyme | Sigma-Aldrich | L6876 | |
| Cytochrome C | Sigma-Aldrich | C7752 | |
| Myoglobin | Sigma-Aldrich | M1882 | |
| ß-casein | Sgma-Aldrich | C6905 | |
| Carbonic anhydrase | Sigma-Aldrich | C3934 | |
| Bovine serum albumin | Sigma-Aldrich | A2153 | |
| Urea | Alfa Aesar | 36428-36 | |
| DL-Dithiothreitol | Sigma-Aldrich | D0632 | Health Hazard |
| Iodoacetamide | Fisher Scientific | AC122270250 | Health Hazard |
| Formic Acid | Fisher Scientific | A117-50 | Corrosive, Health Hazard |
| C4 trap column | Sepax Technologies | 110043-4001C | 3 µm particles, 300 Å pores, 4.0 mm i.d. 10 mm long |
| Acetonitrile | Fisher Scientific | A998SK-4 | Toxic, Oxidizer |
| Ammonium bicarbonate | Sigma-Aldrich | 1066-33-7 | |
| Nalgene rapid-flow filters | Thermo Scientific | 126-0020 | 0.2 µm CN membrane, and 50 mm diameter |
| E. coli cells | K-12 MG1655 | ||
| Dulbecco's phosphate-buffered saline | Sigma-Aldrich | D8537 | |
| BCA assay | Thermo Scientific | 23250 | |
| Acetone | Fisher Scientific | A11-1 | |
| HPLC system for protein desalting | Agilient | 1260 Infinity II | |
| Acetic Acid | Fisher Scientific | A38-212 | |
| CE autosampler | CMP Scientific | ECE-001 | |
| Electro-kinetically pumped sheath flow interface | CMP Scientific | ||
| Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer | Thermo Fisher Scientific | ||
| Sutter flaming/brown micropipette puller | Sutter Instruments | P-1000 | |
| Ultrasonic cell disruptor for cell lysis | Branson | 101063196 | Model S-250A |
| Vaccum concentrator | Thermo Fisher Scientific | SPD131DDA-115 |
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