Global Measurements of Protein Folding Stability for Characterization of Aging and Disease

摘要 基于基因组和蛋白质组的基因表达谱研究已被广泛应用 在过去的十年里，为了表征生物状态，包括那些与正常相关的状态 生物过程(如衰老)和疾病(如癌症)。而这种差异基因 表达谱研究有助于确定细胞通路和生理过程 与一种生物状态相关联，他们往往不能完全理解 生物状态，因为基因表达水平与蛋白质功能没有直接联系。这有 限制了从这些生物标记物中发现的有用蛋白质生物标记物和治疗靶点的数量 研究，并在我们对疾病状态的理解中留下了空白。在这里提出的是发展 以及应用蛋白质稳定性的热力学测量来表征不同 生物状态，包括那些与衰老和癌症相关的状态。这样的热力学 蛋白质稳定性的测量预计将与蛋白质功能更密切地相关 从而产生更多有用的蛋白质生物标志物和 疾病的治疗靶点，并产生更好的理解分子基础 疾病。 拟议的工作将进一步开发和利用三个基于质谱学的 蛋白质组学方法，称为SPROX，LIP和PP，以表征蛋白质折叠和 与基本的生物过程、衰老和疾病相关的稳定性变化， 结直肠癌。这项工作的具体目标是：(1)将SPROX和LIP与中间层连接起来。 羽绒蛋白质组学方法使蛋白质形式特异性折叠和稳定性测量成为可能；(2) 利用(1)中开发的中间向下SPROX和LIP工作流制作Proteoform 小鼠脑蛋白的特殊热力学稳定性测定 衰老模型；(3)利用SPROX、LIP和PP技术鉴定蛋白质生物标志物 不同的患者来源的结直肠癌细胞系的折叠和稳定性发生变化 对奥沙利铂治疗的敏感性；(4)利用(3)中发现的蛋白质生物标志物 建立一种预测奥沙利铂耐药/敏感性的临床相关方法 癌症。

ABSTRACT Genome- and proteome-based gene expression profiling studies have been widely used over the past decade to characterize biological states including those associated with normal biological process (e.g., aging) and with disease (e.g., cancer). While such differential gene expression profiling studies can help identify the cellular pathways and physiological processes associated with a biological state they often fail to produce a complete understanding of the biological state because gene expression levels are not directly tied to protein function. This has limited the number of useful protein biomarkers and therapeutic targets discovered from such studies, and left gaps in our understanding of disease states. Proposed here is the development and application of thermodynamic measurements of protein stability to characterize different biological states including those associated with aging and cancer. Such thermodynamic measurements of protein stability are expected to be more closely related to protein function than are protein expression levels, and thus produce more useful protein biomarkers and therapeutic targets of disease and generate a better understanding of the molecular basis of disease. The proposed work will further develop and utilize three mass spectrometry-based proteomics methods, termed SPROX, LiP and PP, to characterize the protein folding and stability changes associated with a fundamental biological process, aging, and a disease, colorectal cancer. The specific aims of this work are: (1) to interface SPROX and LiP with middle- down proteomics methods to enable proteoform specific folding and stability measurments; (2) to utilize the middle down SPROX and LiP workflows developed in (1) to make proteoform specific thermodynamic stability measurements on mouse brain proteins derived from a mouse model of aging; (3) to utilize the SPROX, LiP, and PP techniques to identify protein biomarker with altered folding and stability in patient-derived colorectal cancer cell lines with different sensitivities to oxaliplatin treatment; (4) to utilize the protein biomarkers discovered in (3) to develop a clinically relevant assay for predicting oxaliplatin resistance/sensitivity in colorectal cancer.