PTM MAPPING IN HUMAN H-RAS UNDER OXIDATIVE STRESSES

氧化应激下人类 H-RAS 的 PTM 作图

• 批准号: 8365567
• 负责人: RICHARD A COHEN
• 金额: $ 1.85万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365567
• 关键词: Adenoviruses; Biology; Cell Line; Cell Proliferation; Cell membrane; Cleaved cell; Cysteine; Dialysis procedure; Digestion; Endothelial Cells; Fourier transform ion cyclotron resonance; Funding; Gel; Grant; Guanosine Triphosphate Phosphohydrolases; HRAS gene; High Pressure Liquid Chromatography; Human; Magnetism; Maps; Mass Spectrum Analysis; Medicine; Methods; Modification; National Center for Research Resources; Oxidative Stress; Peptides; Polyethylene Glycols; Post-Translational Protein Processing; Principal Investigator; Proteins; Protocols documentation; Recovery; Research; Research Infrastructure; Resources; Sampling; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; United States National Institutes of Health; cost; interest; protein function; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. H-Ras is a plasma membrane associated GTPase, which is an important player in signal transduction to control cell proliferation, differentiation, and invasion. There are four reactive cysteines (C118, C181, C184 and C186) on H-Ras. These cysteines are targets of posttranslational modifications (PTM) which may alter the cellular localization and function of this protein. In this study, PTMs on H-Ras in human aortic endothelial cells (HAECs) is being investigated by mass spectrometry to obtain the full map of H-Ras modifications under different oxidative stresses. H-Ras is not an abundant protein that can be easily extracted from cell lines. A protocol has been developed to facilitate the enrichment and purification of the H-Ras, where exogenous H-Ras with 6X His tag was transduced by adenovirus, and pulled down by magnetic Ni-NTA beads. Tryptic in-gel digestion was performed on the His-tagged H-Ras, followed by MS analysis using both MALDI-TOF and LTQ-Orbitrap. Although high sequence coverage was obtained by combining these two methods, only C181 and C184 containing peptides were identified in the mass spectrum. The C186 containing tryptic peptide CVLS was absent in either experiment, because VLS is known to be cleaved after C186 gets farnesylated. Since two cysteine residues of interest were not covered in the bottom-up analysis, a top-down approach by Fourier-transform ion cyclotron resonance (FT-ICR) MS is now being developed. Several methods have been tried for the purification of the H-Ras sample, including those that use the Ziptip (C4 and C18) or centrifugal filters, dialysis, and the CapLC. It was found that the combination of dialysis and use of the Poros 50 R1 column provides the best result with the highest protein recovery yield and least interference from polyethylene glycol (PEG) and other undesirable contaminants. HPLC and Michrom desalting trap will be tried next.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 H-Ras 是一种质膜相关 GTP 酶，在控制细胞增殖、分化和侵袭的信号转导中发挥着重要作用。 H-Ras 上有四个反应性半胱氨酸（C118、C181、C184 和 C186）。这些半胱氨酸是翻译后修饰 (PTM) 的目标，可能会改变该蛋白质的细胞定位和功能。在本研究中，通过质谱法研究人主动脉内皮细胞 (HAEC) 中 H-Ras 的 PTM，以获得不同氧化应激下 H-Ras 修饰的完整图谱。 H-Ras 不是一种可以轻易从细胞系中提取的丰富蛋白质。已经开发了一种协议来促进 H-Ras 的富集和纯化，其中带有 6X His 标签的外源 H-Ras 通过腺病毒转导，并通过磁性 Ni-NTA 珠拉下来。对带有 His 标签的 H-Ras 进行胰蛋白酶凝胶消化，然后使用 MALDI-TOF 和 LTQ-Orbitrap 进行 MS 分析。尽管结合这两种方法获得了高序列覆盖率，但在质谱中仅鉴定出含有 C181 和 C184 的肽。两个实验中均不存在含有胰蛋白酶肽 CVLS 的 C186，因为已知 VLS 在 C186 法尼基化后会被裂解。 由于自下而上的分析未涵盖两个感兴趣的半胱氨酸残基，因此目前正在开发一种采用傅立叶变换离子回旋共振 (FT-ICR) MS 的自上而下的方法。已经尝试了多种方法来纯化 H-Ras 样品，包括使用 Ziptip（C4 和 C18）或离心过滤器、透析和 CapLC 的方法。结果发现，透析与 Poros 50 R1 色谱柱的结合使用可提供最佳结果、最高的蛋白质回收率以及最少的聚乙二醇 (PEG) 和其他不良污染物的干扰。接下来将尝试HPLC和Michrom脱盐阱。