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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 H-Ras是一种质膜相关的GT3，在细胞增殖、分化和侵袭的信号转导中起重要作用。在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）的靶标，其可以改变该蛋白质的细胞定位和功能。在这项研究中，PTMs对H-Ras在人主动脉内皮细胞（HAECs）正在研究通过质谱法获得不同的氧化应激下的H-Ras修饰的完整地图。 H-Ras不是一种可以容易地从细胞系中提取的丰富蛋白。已经开发了一种方案来促进H-Ras的富集和纯化，其中具有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。对His标记的H-Ras进行胰蛋白酶凝胶内消化，然后使用MALDI-TOF和LTQ-Orbitrap进行MS分析。虽然通过结合这两种方法获得了高的序列覆盖率，但在质谱中仅鉴定出含有C181和C184的肽。在任一实验中不存在含有C186的胰蛋白酶肽CVLS，因为已知VLS在C186被法尼基化后被切割。 由于两个半胱氨酸残基的利益是不包括在自下而上的分析，一个自上而下的方法傅里叶变换离子回旋共振（FT-ICR）MS现在正在开发。已经尝试了几种方法来纯化H-Ras样品，包括使用Ziptip（C4和C18）或离心过滤器、透析和CapLC的那些。结果发现，透析和使用波罗斯50 R1色谱柱的组合提供了最佳结果，具有最高的蛋白质回收率和最少的聚乙二醇（PEG）和其他不良污染物干扰。接下来将尝试HPLC和Michrom反相器。