Quantitative Characterization of Glycopeptide Isomers

由于迫切需要全面了解糖基化的生物学属性 许多关键的生物学功能，如免疫反应、细胞发育、细胞 分化/黏附和宿主-病原体的相互作用，糖蛋白质组学继续是一个高度动态的 研究领域。几十年来，异常糖基化被认为是许多哺乳动物的属性 疾病，包括骨关节炎、囊性纤维化和癌症。此外，糖蛋白的异构体变化 已经在阿尔茨海默氏症和癌症等疾病中观察到。近年来，葡聚糖及其异构体 据报道，它们对SARS-CoV-2病毒感染至关重要，使其成为药物的关键靶点 发展新冠肺炎。因此，糖肽及其异构体的可靠和有效的表征 对于更好地理解生物和生物医学过程中糖基化的属性是必要的。我们是 提出四个具体目标：目标1.加强糖肽异构体的分离和鉴定 利用介孔石墨化碳(MGC)-LC-MS和多肽疏水性指数(HIP)； 利用15N代谢-TMT多路技术和A 平行反应监测(PRM)方法；目的3.加强糖肽同分异构体的表征 新的衍生化方法；以及目标4.通过 改进软件的开发。这些目标的结果将提供可靠和有效的糖蛋白组学。 用于糖肽更好的异构体表征的平台和算法可用于 解决生物医学问题，从而为糖科学社区做出贡献。这项建议的创新之处 这源于所提议的分析方法和软件的独特性。异构体的分离 利用MGC-LC-MS/MS的糖肽是我们实验室开发的一种高度创新的方法，允许高效 糖肽异构体在1厘米短柱上的分离。葡聚糖在体内的保留时间归一化 实验室之间的相互作用已经被引入，并在以前通过葡萄糖单位被证明是必要的 Index(图形用户界面)，但不在糖蛋白质组分析中。我们将首次调查保留时间 用一组已知的多肽对糖肽和糖肽异构体进行归一化 不同仪器和不同实验室的疏水性因素。15N稳定同位素的组合 体外研究中，糖肽和TMT的标记将使TMT的多重能力增加一倍，达到36-plex 细胞系糖蛋白组学。虽然PRM已被用于糖蛋白组谱分析，但异构体分析 缺乏糖蛋白组学。因此，这将是首次对糖肽异构体进行全面研究。 PRM。此外，我们提出的衍生化方法具有很大的创新性。这将是第一次实现 2-氨基苯甲酰胺(2-AB)标记糖肽的唾液酸，并首次实现高效异构体 通过两步氧化还原胺化反应在15 cm C18色谱柱上分离唾液酸糖多肽。 此外，我们还实现了唾液酸化糖肽的衍生化(DOSG)方法，该方法将引入 用质量差异区分α2，3和α2，6连接的唾液酸糖多肽。因此，组合氧化- 还原胺化和DOSG方法导致了唾液酸糖多肽的一种创新的富集法。这个 糖链和氨基酸上唾液酸羧基的衍生化中和 额外的电荷和季铵盐官能化分子的添加提供了可控的 正电荷使得使用强阳离子交换(SCX)进行富集化成为可能。这是一部小说 一种可区分唾液酸键合异构体的高效富集唾液酸糖多肽的方法 在同一时间。该提案的可交付内容是可靠、适应性强且负担得起的战略和 改进的软件，以加强任何有兴趣定义的实验室的异构体糖肽研究 利用LC-MS/MS进行全面的蛋白质糖基化。 更好地理解糖蛋白异构体在肿瘤发生发展中的生物学属性 疾病。

Because of the pressing needs to comprehensively understand the biological attributes of glycosylation in many critical biological functions such as the immune response, cell development, cellular differentiation/adhesion and host-pathogen interactions, glycoproteomics continues to be a highly dynamic research area. Aberrant glycosylation for decades has been recognized as the attribute of many mammalian diseases, including osteoarthritis, cystic fibrosis, and cancer. Moreover, isomeric alterations of glycoproteins have been observed in diseases such as Alzheimer’s Disease and cancers. Recently, glycans and their isomers have been reported to be vital to the SARS-CoV-2 viral infection, making them a crucial target for the drug development of COVID-19. Therefore, reliable, and efficient characterization of glycopeptides and their isomers is necessary to better understand the attributes of glycosylation in biological and biomedical processes. We are proposing here four specific aims: Aim 1. To enhance the separation and identification of glycopeptide isomers using mesoporous graphitized carbon (MGC)-LC-MS and hydrophobicity index of peptides (HIP); Aim 2. To enhance the quantification of glycopeptide isomers using a 15N metabolic-TMT multiplexing approach and a parallel reaction monitoring (PRM) method; Aim 3. To enhance the glycopeptide isomeric characterization using novel derivatization methods; and Aim 4. To enhance automated isomeric glycopeptide data processing by the development of improved software. The outcome of these aims will provide reliable and efficient glycoproteomic platforms and algorithms for a better isomeric characterization of glycopeptides which can be employed to address biomedical issue, thus contributing to the glycoscience community. The innovations of this proposal originate from the uniqueness of the proposed analytical methods and software. The isomeric separation of glycopeptides using MGC-LC-MS/MS is a highly innovative method, developed in our lab, permitting efficient separation of glycopeptide isomers on a 1 cm short column. The retention time normalization of glycans intra and inter-laboratories has been introduced and demonstrated to be necessary previously through a glucose unit index (GUI), but not in glycoproteomic analysis. For the first time, we will investigate the retention time normalization of glycopeptides and glycopeptide isomers using a set of peptides which have known hydrophobicity factors on different instruments and different laboratories. The combination of 15N stable isotope labeling of glycopeptides and TMT will double the multiplexing capacity of TMT to 36-plex when studying in vitro cell line glycoproteomics. Although PRM has been utilized for glycoproteomic profiling, the analysis of isomeric glycoproteomics is lacking. Thus, this will be the first comprehensive investigation of glycopeptide isomers using PRM. Moreover, the derivatization methods we proposed are of great innovation. It will be the first time to achieve 2-aminobenzamide (2-AB) labeling on sialic acids of glycopeptides, and the first time to achieve efficient isomeric separation of sialoglycopeptides on a 15 cm C18 column via a two-step oxidation-reductive amination reaction. In addition, we have achieved a derivatization of sialylated glycopeptides (DOSG) method that will introduce mass difference to distinguish α2,3 and α2,6 linked sialoglycopeptides. Consequently, the combination oxidation- reductive amination and DOSG methods leads to an innovative enrichment method for sialoglycopeptides. The derivatization of carboxyl groups of sialic acids on glycans and amino acids on peptide backbone neutralizes additional charges and the addition of quaternary ammonium functionalized molecules provides controllable positive charges which make the enrichment using strong cation exchange (SCX) possible. This is a novel method for the efficient enrichment of sialoglycopeptides where sialic acid linkage isomers can be distinguished at the same time. The deliverables of this proposal are reliable, adaptable, and affordable strategies and improved software to enhance the isomeric glycopeptide studies by any laboratory interested in defining comprehensive protein glycosylation using LC-MS/MS. The proposed technologies are expected to enable a better understanding of the biological attributes of glycoprotein isomers in the development and progression of diseases.