Site-Specific Analysis of Human Cysteine Sulfenic Acid Protein Modifications

描述（由申请人提供）：生物医学科学家需要不断改进的工具来面对理解蛋白质调控及其与疾病状态关系的复杂挑战。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调节蛋白质活性的一种手段，有助于吸收、信息记录（为了激活生物反馈机制的目的）和/或改变蛋白质活性，并转移否则可能是有害的侮辱。半胱氨酸磺酸的形成是一种特别难以追踪的修饰（由于其固有的茧样蛋白质环境之外的不稳定性），似乎在许多蛋白质的生化调节中起着关键作用。这种蛋白质修饰的分析难以捉摸的性质使得了解其生物学作用，从而了解任何受影响蛋白质的全部生物学作用，这是一个经常被忽视的优先事项。该应用的驱动假设是，通过半胱氨酸磺酸形成的蛋白质的氧化[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的蛋白质的活性[即具有游离巯基官能团的半胱氨酸残基]。因此，本研究的长期目标是开发一种方法，促进氨基酸残基特异性鉴定和定量半胱氨酸磺酸修饰的特定靶蛋白直接脱离其生物环境，如可能适合于临床诊断。这一目标将通过完成三个特定目标来实现：1)合成一种定制设计的三功能分子标签，可用于特异性标记蛋白质中半胱氨酸磺酸的位点，同时为亲和纯化提供处理，并在初步分析（单级质谱）中提供标记模式，表明所标记肽的存在和分子质量。2)优化血浆样品制备，对特异性靶蛋白内的半胱氨酸磺化事件进行定性鉴定和相对定量。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，规范未氧化的[含游离硫醇]蛋白质，用于半胱氨酸磺酸修饰。

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.