Analytical and Informatic Approach to Determine Disease-related Glycoforms of Proteins

摘要 与疾病相关的蛋白质糖基化改变的生物医学意义已被公认为 过段时间吧。然而，在将这些观察结果转化为强大的疾病方面仍然存在重大挑战。 生物标志物，以及建立对改变的蛋白质糖基化如何影响的详细分子理解 多种生化相互作用和信号通路，包括蛋白质-药物相互作用。此外， 因为蛋白质糖基化是一系列复杂的非模板驱动的生物合成和 后处理步骤，蛋白质糖基化图谱是复杂的生物分子网络的产物 相互作用，因此对代谢信号通路中的扰动非常敏感，包括 伴随疾病。由于这些原因，蛋白质糖基化的详细分子特征是 对疾病的生物标记物的发现以及与疾病相关的生物医学的极大兴趣 蛋白质糖基化对蛋白质功能的影响。这种努力的关键是强大的分析能力。 可以确定蛋白质连接的寡糖的组成和结构的技术及其 蛋白质在复杂和非均相混合物中的附着位置。针对特定站点的中心工具 蛋白质糖基化的表征是串联质谱学(MS/MS)在 糖蛋白通过蛋白质分解消化而产生的糖肽。这类分析的一个非常理想的结果 是获得有关寡糖拓扑结构和多肽序列的信息。目前，这个 通常，只有通过组合多种MS/MS方法才能达到详细程度，代价是速度和 表征的糖肽的数量。为了克服这一障碍，我们将开发一种彻底的化学物质 了解最常见的MS/MS方法--碰撞诱导解离(CID)--如何单独 提供N-连接糖肽的完整连接性信息。我们将开发和应用这一点 1)确定了电荷状态、单糖组成、氨基酸等因素的影响 糖苷和多肽的组成和振动自由度对CID能量的要求 质子化N-糖肽中的键断裂；2)编辑和挖掘一个大型的CID谱数据库 不同碰撞能下的质子化N-糖肽对前体离子特性的经验关联 具有依赖能量的解离特性；以及3)通过以下方式说明这些方法和见解 检测人α1-酸性糖蛋白(AGP)位点特异性N-糖基化的变化 发生在类风湿性关节炎期间。这些目标的实现将由双方的能力来实现 系统生物学核心设施和内布拉斯加州中心的数据管理和分析核心设施 集成生物分子通信(CIBC)用于1)获取大型高维数据集，以及2)应用 以信息学方法挖掘这些数据集，以获得对糖肽解离的见解。这些洞察力将是 直接适用于疾病引起的特定部位糖基化模式的阐明。

ABSTRACT The biomedical significance of disease-related alterations in protein glycosylation has been recognized for some time. However, significant challenges remain in translating these observations into robust disease biomarkers, and in establishing a detailed molecular understanding of how altered protein glycosylation affects a variety of biochemical interactions and signaling pathways, including protein-drug interactions. Moreover, because protein glycosylation is the end result of a complex series of non-template-driven biosynthetic and post-processing steps, a protein glycosylation profile is the product of an intricate network of biomolecular interactions and is thus exquisitely sensitive to perturbations in metabolic signaling pathways, including those accompanying disease. For these reasons, the detailed molecular characterization of protein glycosylation is of great biomedical interest in biomarker discovery for diseases and for the purpose of relating disease-induced alterations in protein glycosylation to protein function. Essential to such endeavors are powerful analytical techniques that can determine the compositions and structures of protein-linked oligosaccharides and their sites of protein attachment in complex and heterogeneous mixtures. A central tool for the site-specific characterization of protein glycosylation is the application of tandem mass spectrometry (MS/MS) to glycopeptides produced by proteolytic digestion of glycoproteins. A highly desirable outcome of such analyses is to obtain information on both the oligosaccharide topology and the polypeptide sequence. At present, this level of detail is typically achieved only by combining multiple MS/MS methods at the expense of speed and number of glycopeptides characterized. To overcome this barrier, we will develop a thorough chemical understanding of how the most common MS/MS method - collision-induced dissociation (CID) - can alone provide complete connectivity information on N-linked glycopeptides. We will develop and apply this understanding by: 1) establishing the influence of charge state, monosaccharide composition, amino acid composition, and vibrational degrees of freedom on the CID energy requirements for glycosidic and peptide bond scission in protonated N-glycopeptides; 2) compiling and mining a large database of CID spectra of protonated N-glycopeptides at various collision energies to empirically correlate precursor ion characteristics with energy-dependent dissociation characteristics; and 3) illustrating these methods and insights by examining changes in the site-specific N-glycosylation profile of human alpha1-acid glycoprotein (AGP) that occur during rheumatoid arthritis. Achievement of these aims will be enabled by the capabilities of both the Systems Biology Core facility and the Data Management and Analysis Core facility of the Nebraska Center for Integrated Biomolecular Communication (CIBC) to 1) obtain large, high-dimensional data sets, and 2) apply informatic methods to mine these data sets for insights on glycopeptide dissociation. These insights will be directly applicable to the elucidation of site-specific glycosylation patterns that arise as a result of disease.