蛋白酶和酸催化的贴标工作流程，用人<sup> 18</sup> O-富氧水
Summary
稳定同位素标记的工作流程，采用<sup> 18</sup> O-富氧水（LEO-工作流）的多用途工具进行定量和定性的蛋白质组学研究。在蛋白酶辅助（古）的工作流程，<sup> 18</sup> O原子的引入，通过蛋白水解和羧基氧交换反应介导的蛋白酶。在酸催化（ALEO）工作流程中，<sup> 18</sup> O原子在低pH条件下引入羧基氧交换。
Abstract
稳定同位素在生物质谱技术是必不可少的工具。从历史上看，稳定同位素18 O已被广泛用来研究蛋白水解酶1-3的催化机制。随着质谱为基础的蛋白质组学，酶催化成立18 O原子从稳定同位素富集的水已经成为一种流行的方法来定量比较蛋白质表达水平（由Fenselau和姚明，宫城县和4饶5和叶审查等 。6），18 O标记构成一个简单的，低成本的替代化学品（ 如 iTRAQ的，ICAT）和代谢标记技术（ 如 SILAC）。根据使用的蛋白酶的18 O-标记可以导致将多达两个18 O-原子中的C-末端羧基基团的裂解产物3 </sup>。标记反应可以被细分成两个独立的过程，在肽键裂解和羧基氧交换反应8。在我们的古（P rotease 一个的 ssisted的升abelingËmploying 18 O-富氧水）改编酶18 O标记，我们采用50％的18 O富集的水，产生独特的同位素特征。结合高分辨率基质辅助激光解吸电离飞行时间飞行串联质谱（MALDI-TOF/TOF MS / MS），特征同位素信封可以用来识别与高水平的特异性的裂解产品。我们以前使用过的古方法来检测和表征内源性蛋白酶9和监控蛋白水解反应10-11。 ，由于古编码的蛋白裂解反应的本质，实验装置简单，生化ENRIchment步骤裂解产物可以被规避。古方法可以很容易地扩展到（I）课程实验，监测蛋白水解裂解反应的动力学及（ii）分析在复杂的生理条件下的生物样本，分别代表水解。古时间过程的实验有助于确定限速的处理步骤和反应中间体，在复杂的蛋白水解途径反应。此外，古反应，使我们能够识别蛋白水解酶的丝氨酸蛋白酶如胰蛋白酶，能够重新绑定其裂解产物，并促进成立的第二个18 O原子。这种“双标记”酶可用于postdigestion 18 O标记，其中肽是专门标记的羧基氧交换反应。我们的第三个战略扩展标记采用18 O富集的水以外的酶，并使用酸性的pH值条件下，引进18 O稳定同位素标志atures肽。
Protocol
所提出的LEO工作流允许的稳定同位素标记的蛋白质消化和合成肽。这些课程实验（ 图1）是适用于比较和定量蛋白质组学的研究，以及蛋白酶的研究。每个工作流程由两个实验的步骤（ 图2）：A）各自的18 O-稳定的同位素编码的反应（蛋白酶催化肽切割蛋白酶催化羧基氧交换反应，酸催化的羧基氧交换的时间分辨的采样反应）和B）通过质谱法和图形表示的18 O</…
Representative Results
我们使用动态监控结合成肽蛋白水解酶裂解产生18 O稳定同位素的古时间过程的工作流程。所提出的方法是一种多功能的工具，不同的底物和蛋白酶的组合比较研究蛋白水解处理途径。通过重复采样蛋白水解反应，在反应过程中的，古时间过程的时间分辨实验提供了快照的底物和产物的丰度和处理的详细信息。共发现样本与目标板的酸性基质溶液停止酶的反应和基体结晶，进一步保证样品?…
Discussion
结合稳定同位素标记和高分辨率质谱在时间分辨的方式，在古时间过程的方法允许产生的肽产品的动态分析。该试剂盒可用于产生稳定的同位素标记的肽进行定量和定性的蛋白质组学研究来评估肽段生成的动力学。此外，古时间过程被设计来评估，根据特定的，生理上有关的条件体外水解途径，可以利用工作流中的内源的蛋白质，肽和蛋白酶，以及合成肽和重组蛋白酶。根据特定的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是支持由NIH / NIDCR的格兰特1R01DE019796。
Materials
| Name of material | Company | Catalogue number |
| PepClean C-18 Spin Columns | Thermo | 89870 |
| Opti-TOF 384 MALDI target plate | AB SCIEX | 1016629 |
| 4800 MALDI TOF/TOF | AB SCIEX |
Table 1. Materials
| Name of reagent | Company | Catalogue number |
| Alpha cyano-4-hydroxycinnamic acid | Sigma Aldrich | 70990-1G-F |
| Bovine serum albumin (BSA) | Sigma Aldrich | A3294-10G |
| Dithiothreitol (DTT) | Acros | 16568-0050 |
| Iodoacetamide (IAM) | Sigma Aldrich | 1149-5G |
| Endothelin converting enzyme-1 (ECE-1) | R&D Systems | 1784-ZN |
| Trypsin Gold | Promega | V5280 |
| Water-18O, 97 atom % 18O | Sigma Aldrich | 329878-1G |
| Trifluoroacetic acid (TFA) | Thermo | 28904 |
| Mass Standards Kit for Calibration of AB SCIEX TOF/TOF instruments | AB SCIEX | 4333604 |
Table 2. Reagents
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Tags
Protease-catalyzed LabelingAcid-catalyzed Labeling18O-enriched WaterStable IsotopesBiological Mass SpectrometryCatalytic MechanismsProteolytic EnzymesMass Spectrometry-based ProteomicsProtein Expression Levels18O-labelingPeptide Bond CleavageCarboxyl Oxygen Exchange ReactionPALeO AdaptationEnzymatic 18O-labelingDistinctive Isotope SignaturesHigh-resolution Matrix-assisted Laser Desorption Ionization Time-of-flight Tandem Mass Spectrometry (MALDI-TOF/TOF MS/MS)Isotope Envelopes
Citar este artigo
Klingler, D., Hardt, M. Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water. J. Vis. Exp. (72), e3891, doi:10.3791/3891 (2013).