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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 H-RAS是质膜相关的GTPase，它是控制细胞增殖，分化和侵袭的信号转导的重要参与者。 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，可能会改变该蛋白的细胞定位和功能。在这项研究中，正在研究人主动脉内皮细胞（HAEC）的H-RAS上的PTMS通过质谱研究，以获得不同氧化应激下H-RAS修饰的完整图。 H-RAS不是可以轻松从细胞系中提取的丰富蛋白质。已经开发了一项方案，以促进H-RAS的富集和纯化，在该方案中，外源H-Ras用6倍的标签被腺病毒转导，并被磁性Ni-NTA珠子拉下。在His标记的H-RAS上进行了胰蛋白酶内消化，然后使用MALDI-TOF和LTQ-ORBITRAP进行MS分析。尽管通过组合这两种方法获得了高序列覆盖率，但在质谱中仅鉴定了含有肽的C181和C184。在任何一个实验中都不存在含有胰蛋白酶肽CVL的C186，因为已知VLS在C186被裂解后被裂解。 由于自下而上的分析未涵盖两种感兴趣的半胱氨酸残基，因此现在正在开发傅立叶转换离子回旋共振（FT-ICR）MS的自上而下方法。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的方法。发现透析和使用Poros 50 R1色谱柱的结合提供了最佳的结果，其蛋白质回收率最高，并且来自聚乙烯乙二醇（PEG）（PEG）和其他不良污染物的干扰最少。接下来将尝试HPLC和MICHROM脱盐陷阱。