PTM MAPPING IN HUMAN H-RAS UNDER OXIDATIVE STRESSES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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。

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.