Site-Specific Analysis of Human Cysteine Sulfenic Acid Protein Modifications

人半胱氨酸磺酸蛋白修饰的位点特异性分析

• 批准号: 7347362
• 负责人: CHAD R BORGES
• 金额: $ 18.57万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347362
• 关键词: Acceleration; Affect; Affinity; Affinity Chromatography; Alkylation; Amino Acids; Automobile Driving; Avidin; Biochemical; Biochemistry; Biological; Biological Process; Biological feedback; Biotin; Cellular biology; Characteristics; Chemicals; Chemistry; Collection; Complex; Condition; Custom; Cysteine; Data; Development; Disease; Disulfides; Endopeptidases; Environment; Ethylmaleimide; Event; Exanthema; Face; Fingerprint; Funding; Generations; Goals; Human; Immunoassay; Ions; Isotopically-Coded Affinity Tagging; Label; Lasers; Light; Maleimides; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Modification; Molecular; Molecular Probes; Morphologic artifacts; Nature; Numbers; Oxidation-Reduction; Pattern; Peptide Hydrolases; Peptides; Plasma; Play; Post-Translational Protein Processing; Preparation; Procedures; Protein Biochemistry; Proteins; Proteolysis; Purpose; Range; Reagent; Regulation; Relative (related person); Research; Research Design; Research Personnel; Residual state; Robotics; Role; Sampling; Scanning; Site; Specific qualifier value; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Staging; Streptavidin; Structure; Sulfenic Acids; Sulfhydryl Compounds; Techniques; Technology; Testing; Time; Validation; Variant; base; biomedical scientist; clinical Diagnosis; clinically relevant; cysteinesulfenic acid; day; design; desire; disulfide bond; field study; functional group; high throughput analysis; improved; interest; mass spectrometer; method development; molecular mass; novel; prevent; protein purification; tool; ultraviolet

DESCRIPTION (provided by applicant): Biomedical scientists require ever improving tools to face the complex challenge of understanding the intricacies of protein regulation and its relationship to disease states. Recent research in the field of protein biochemistry has revealed that in many cases oxidative chemical modifications to protein molecules actually serve as a means of regulating protein activity helping to absorb, informationally register (for the purpose of activating biological feedback mechanisms) and/or alter protein activity, and deflect what otherwise might have been injurious insults. Cysteine sulfenic acid formation stands as one such particularly difficult-to-track modification (due to its inherent instability outside of its native cocoon-like protein environment) that appears to play a key role in the biochemical regulation of many proteins. The analytically elusive nature of this protein modification has made understanding its biological role and thus the full biological role of any affected protein an oft-ignored priority. The driving hypothesis of this application is that oxidative [redox] regulation of proteins via cysteine sulfenic acid formation critically affects the activity of proteins containing one or more unoxidized cysteine residues [i.e., cysteine residues with free thiol functional groups]. The long term goal of this research is, thus, to develop methodology that facilitates the amino acid residue-specific identification and quantitation of cysteine sulfenic acid modifications of specific target proteins directly out of their biological environment such as might be appropriate for making clinical diagnoses. This goal will be realized through completion of three specific aims: 1) Synthesize a custom-designed trifunctional molecular tag that can be used to specifically label site(s) of cysteine sulfenic acid within proteins and simultaneously provide a handle for affinity purification and a signature pattern in preliminary analyses [within single stage mass spectra] that indicates the presence and molecular mass of labeled peptides. 2) Optimize the preparation of blood plasma samples for qualitative identification and relative quantitation of cysteine sulfenation events within specifically targeted proteins. And 3) validate preparatory and analytical procedures according to the Association of Analytical Chemists-recommended method validation criteria and begin to qualitatively and quantitatively test biologically extracted, genuinely unknown, normatively unoxidized [free thiol-containing] proteins for cysteine sulfenic acid modifications. Recent evidence shows that within proteins, a uniquely modified (oxidized) form of the amino acid cysteine known as cysteine sulfenic acid can play important regulatory roles in both day-to-day biological function and in disease states. Because of their unstable nature, however, the biologically important details of such modifications go all but unnoticed under traditional techniques used to directly analyze proteins. The project under proposal here aims to remedy this analytical deficit by providing a robust means to study the detailed qualitative and quantitative nature of cysteine sulfenic acid modifications in proteins extracted directly from clinically relevant biological samples.

描述(申请人提供)：生物医学科学家需要不断改进的工具来面对复杂的挑战，即理解复杂的蛋白质调节及其与疾病状态的关系。最近在蛋白质生物化学领域的研究表明，在许多情况下，对蛋白质分子的氧化化学修饰实际上是一种调节蛋白质活性的手段，有助于吸收、信息登记(目的是激活生物反馈机制)和/或改变蛋白质活性，并转移本来可能是有害侮辱的东西。半胱氨酸磺酸的形成是一种特别难以追踪的修饰(由于其天然的类茧蛋白环境之外的固有不稳定性)，似乎在许多蛋白质的生化调节中发挥关键作用。这种蛋白质修饰的分析难以捉摸的性质使得理解它的生物学作用，从而使任何受影响的蛋白质的全部生物学作用成为经常被忽视的优先事项。这一应用的驱动假设是，通过形成半胱氨酸对蛋白质的氧化[氧化还原]调节严重影响含有一个或多个未氧化半胱氨酸残基(即具有自由硫醇官能团的半胱氨酸残基)的蛋白质的活性。因此，这项研究的长期目标是开发一种方法学，便于直接从特定目标蛋白的生物环境中对其进行氨基酸残基特异性鉴定和半胱氨酸磺酸修饰的定量，例如可能适合进行临床诊断。这一目标将通过完成三个具体目标来实现：1)合成一种定制设计的三功能分子标签，用于特异性标记蛋白质中半胱氨酸磺酸的位点(S)，同时提供亲和纯化的句柄和初步分析[在单级质谱图内]指示标记多肽的存在和分子质量的特征模式。2)优化血浆样品的制备，以定性鉴定和相对定量特定目标蛋白内的半胱氨酸磺化事件。3)根据分析化学家协会推荐的方法验证标准验证准备和分析程序，并开始定性和定量测试生物提取的、真正未知的、正常未氧化的[含游离硫醇]蛋白质的半胱氨酸磺酸修饰。 最近的证据表明，在蛋白质中，半胱氨酸的一种独特的修饰(氧化)形式，即半胱氨酸亚磺酸，可以在日常生物功能和疾病状态中发挥重要的调节作用。然而，由于其不稳定的性质，在用于直接分析蛋白质的传统技术下，这种修饰的生物学重要细节几乎不被注意到。建议中的项目旨在通过提供一种强有力的手段来研究直接从临床相关生物样本中提取的蛋白质中半胱氨酸磺酸修饰的详细定性和定量性质，从而弥补这一分析缺陷。