Antibody Glycosylation by Mass Spectrometry

抗体（Abs）是免疫系统中最重要的糖蛋白，其中免疫球蛋白G （IgG）是最常见的代表。igg有许多来源，并且有几种动机来表征它们的糖基化模式和氨基酸（AA）序列，这是本提案的中心主题。PI实验室在糖基化分析方面有20多年的专业经验，培养了许多HQP。该计划涉及至少10名HQP，为期5年，每个HQP都将受益于生物分析化学和质谱（MS）的跨学科培训。建议的各节摘要如下。*******部分IgG糖基化发生在分子的抗原结合区（Fab结构域），不同类型的IgG的AA序列不同。由于文献中关于Fab结构域可变部分的信息很少，因此迫切需要开发可靠的方法和工作流程来表征糖基化和aa方面的Fab。这对于了解更多关于ab抗原相互作用和糖基化在这一过程中的作用是很重要的。* igg将来自三个来源：人类血浆、猪血浆和来自合作者实验室的细胞培养物。*******人血清IgG是多克隆的，即其AA序列不同的亚型（IgG1, IgG2， IgG3和IgG4）。每种亚型对特定受体表现出不同水平的亲和力，但到目前为止，MS很难量化这些亚型。建议开发一种定量方法，该方法将使用每个亚型序列独特的合成肽标准。这四种亚型的比例在人群中略有不同，这种方法将有助于研究免疫系统反应是否与这些比例有关。*******同样有趣的是猪igg。猪和人之间糖基化的差异导致异种移植（从动物移植到人）的问题。正常的猪器官会被人类排斥，这部分是由于蛋白质上的特定末端糖。通过敲除（KO）负责这些的基因，可以为异种移植产生类似人类的器官和液体。目的是充分表征IgG糖基化，以评估KO过程的效率，并了解聚糖在排斥现象中的作用。*******IgG糖基化分析将使用糖蛋白组学方法进行，目前PI实验室正在研究新的捕获-释放方法（可切割连接物），以捕获糖肽并在生物反应器中的单克隆抗体上进行测试。这些新材料由合成在固相材料上的连接剂组成，例如功能化二氧化硅或磁性纳米颗粒。这些产品将引起糖蛋白组学科学界的极大兴趣。******

Antibodies (Abs) are glycoproteins of prime importance in the immune system, with immunoglobulin G (IgG) as their most common representative. There are many sources of IgGs and several motives to characterize their glycosylation patterns and amino acid (AA) sequences, which is the central theme of this proposal. The PI's laboratory has specialized in glycosylation analysis for over 20 years, and has trained many HQP. This proposal involves a minimum of 10 HQP over 5 years and each of them will benefit of interdisciplinary training in bioanalytical chemistry and mass spectrometry (MS). Sections of the proposal are summarized below.*******Part of IgG glycosylation occurs in the antigen-binding region (Fab domain) of the molecule, which varies from one type of IgG to the next with respect to AA sequence. As very little information is available in the literature about the variable portions of Fab domains, there is a strong need for the development of reliable methods and workflows to characterize the Fab with respect to glycosylation and AAs. This is important in order to learn more about Ab-antigen interactions and about the role of glycosylation in this process.*The IgGs will be from three sources: human plasma, swine plasma, and cell cultures from collaborators' laboratories.*******Human serum IgG is polyclonal, i.e. it has subtypes differing in their AA sequences (IgG1, IgG2, IgG3 and IgG4). Each subtype shows different levels affinity for specific receptors, but so far with MS it has been difficult to quantify these subtypes. It is proposed to develop a quantitative method which will use synthetic peptide standards unique to the sequence of each subtype. The ratios of these four subtypes has been found to vary slightly in the population, and this method will help investigate if immune system response may relate to these ratios.*******Also of interest are porcine IgGs. Differences in glycosylation between swine and human result in problems in the context of xenotransplantation (transplantation from animal to human). Normal pig organs are rejected by humans, and this is partly attributed to specific terminal sugars on the proteins. By knocking out (KO) the genes responsible for these, human-like organs and fluids may be generated for xenotransplantation. The objective is to fully characterize IgG glycosylation, in order to assess the efficiency of the KO process and understand the role of glycans in the rejection phenomena.*******IgG glycosylation analyses will be conducted using glycoproteomic approaches, for which novel capture-release methods (cleavable linkers) are presently being investigated in the PI's laboratory for their ability to capture glycopeptides and tested on monoclonal Abs from bioreactors. These new materials consist of linkers synthesized onto solid phase materials, e.g. functionalized silica or magnetic nanoparticles. These products will be of great interest to the glycoproteomics scientific community.******