PTM MAPPING IN HUMAN H-RAS UNDER OXIDATIVE STRESSES
氧化应激下人类 H-RAS 的 PTM 作图
基本信息
- 批准号:8170941
- 负责人:
- 金额:$ 4.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-06-01 至 2011-05-31
- 项目状态:已结题
- 来源:
- 关键词:AdenovirusesCell LineCell ProliferationCell membraneCleaved cellComputer Retrieval of Information on Scientific Projects DatabaseCysteineDialysis procedureDigestionEndothelial CellsFourier transform ion cyclotron resonanceFundingGelGrantGuanosine Triphosphate PhosphohydrolasesHRAS geneHigh Pressure Liquid ChromatographyHumanInstitutionMagnetismMapsMass Spectrum AnalysisMethodsModificationOxidative StressPeptidesPolyethylene GlycolsPost-Translational Protein ProcessingProteinsProtocols documentationRecoveryResearchResearch PersonnelResourcesSamplingSignal TransductionSourceUnited States National Institutes of Healthinterestprotein functionresearch study
项目摘要
This subproject is one of many research subprojects utilizing the
resources provided by a Center grant funded by NIH/NCRR. The subproject and
investigator (PI) may have received primary funding from another NIH source,
and thus could be represented in other CRISP entries. The institution listed is
for the Center, which is not necessarily the institution for the investigator.
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.
这个子项目是众多研究子项目之一
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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RICHARD A COHEN其他文献
RICHARD A COHEN的其他文献
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{{ truncateString('RICHARD A COHEN', 18)}}的其他基金
ID OF OXIDANT SENSITIVE CYSTEINE CONTAINING PROTEINS BY MASS SPECTROMETRY
通过质谱法鉴定含氧化剂敏感半胱氨酸的蛋白质
- 批准号:
8365499 - 财政年份:2011
- 资助金额:
$ 4.05万 - 项目类别:
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 - 财政年份:2011
- 资助金额:
$ 4.05万 - 项目类别:
Redox Regulation of p21ras in Angiogenesis
p21ras 在血管生成中的氧化还原调节
- 批准号:
8109964 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Redox Regulation of p21ras in Angiogenesis
p21ras 在血管生成中的氧化还原调节
- 批准号:
7947453 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Aortic Stiffness and Hypertension in Obese Mice
肥胖小鼠的主动脉僵硬和高血压
- 批准号:
8484428 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Aortic Stiffness and Hypertension in Obese Mice
肥胖小鼠的主动脉僵硬和高血压
- 批准号:
8149954 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Aortic Stiffness and Hypertension in Obese Mice
肥胖小鼠的主动脉僵硬和高血压
- 批准号:
8292165 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Redox Regulation of p21ras in Angiogenesis
p21ras 在血管生成中的氧化还原调节
- 批准号:
8294637 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
Redox Regulation of p21ras in Angiogenesis
p21ras 在血管生成中的氧化还原调节
- 批准号:
8699256 - 财政年份:2010
- 资助金额:
$ 4.05万 - 项目类别:
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