J Am Soc Mass Spectrom. 2022 Oct 5;33(10):1874-1882. doi: 10.1021/jasms.2c00177. Epub 2022 Sep 12.
Protein glycosylation, covalent attachment of carbohydrates to polypeptide chains, is a highly important post-translational modification involved in many essential physiological processes. Comprehensive site-specific and quantitative analysis is crucial for revealing the diverse functions and dynamics of glycosylation. To characterize intact glycopeptides, mass spectrometry (MS)-based glycoproteomics employs versatile fragmentation methods, among which electron-transfer/higher-energy collision dissociation (EThcD) has gained great popularity. However, the inherent limitation of EThcD in fragmenting low-charge ions has prevented its widespread applications. Furthermore, there is a need to develop a high-throughput strategy for comparative glycoproteomics with a large cohort of samples. Herein, we developed isobaric ,-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) tags to increase the charge states of glycopeptides, thereby improving the fragmentation efficiency and allowing for in-depth intact glycopeptide analysis, especially for sialoglycopeptides. Moreover, the unique reporter ions of DiLeuEN-labeled glycopeptides generated in tandem MS spectra enable relative quantification of up to four samples in a single analysis, which represents a new high-throughput method for quantitative glycoproteomics.
蛋白质糖基化是将碳水化合物共价连接到多肽链上的一种高度重要的翻译后修饰，参与许多重要的生理过程。全面的、特异性和定量的分析对于揭示糖基化的多样化功能和动态至关重要。为了对完整糖肽进行特征分析，基于质谱（MS）的糖蛋白质组学采用了多种碎裂方法，其中电子转移/更高能量碰撞解离（EThcD）得到了广泛的应用。然而，EThcD 内在的碎裂低电荷离子的局限性限制了它的广泛应用。此外，需要开发一种用于具有大量样本的比较糖蛋白质组学的高通量策略。在这里，我们开发了等摩尔的、-二甲基亮氨酸衍生的乙二胺（DiLeuEN）标签来增加糖肽的电荷状态，从而提高碎裂效率，并允许对完整糖肽进行深入分析，特别是对唾液酸糖肽。此外，串联质谱图谱中产生的 DiLeuEN 标记糖肽的独特报告离子可以在单次分析中对多达四个样本进行相对定量，这代表了一种新的高通量定量糖蛋白质组学方法。
