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（出于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。半胱氨酸次磺酸的形成是一种特别难以追踪的修饰（由于其在天然茧样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起关键作用。这种蛋白质修饰的分析难以捉摸的性质使得理解其生物学作用以及任何受影响蛋白质的全部生物学作用成为经常被忽视的优先事项。本申请的驱动假设是通过半胱氨酸次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的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方法，该方法有助于直接从其生物环境中对特定靶蛋白的半胱氨酸次磺酸修饰进行氨基酸残基特异性鉴定和定量，例如可能适合于进行临床诊断。这一目标将通过完成三个具体目标来实现：1）合成定制设计的三功能分子标签，其可用于特异性标记蛋白质内的半胱氨酸次磺酸位点，并同时提供亲和纯化的手柄和初步分析中的特征模式[在单级质谱内]，其指示标记肽的存在和分子量。2)优化血浆样品的制备，用于特异性靶向蛋白质内半胱氨酸磺化事件的定性鉴定和相对定量。以及3）根据分析化学家协会推荐的方法验证标准验证制备和分析程序，并开始定性和定量检测生物提取的、真正未知的、正常未氧化的[含游离巯基的]蛋白质的半胱氨酸次磺酸修饰。 最近的证据表明，在蛋白质中，被称为半胱氨酸次磺酸的氨基酸半胱氨酸的独特修饰（氧化）形式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，这种修饰的生物学重要细节在用于直接分析蛋白质的传统技术下几乎被忽视。该项目旨在通过提供一种强大的方法来研究直接从临床相关生物样品中提取的蛋白质中半胱氨酸次磺酸修饰的详细定性和定量性质，从而弥补这种分析缺陷。

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.