Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, the Netherlands; Centre for Analytical Sciences Amsterdam, Science Park 904, 1098 XH, Amsterdam, the Netherlands.
Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, the Netherlands; Centre for Analytical Sciences Amsterdam, Science Park 904, 1098 XH, Amsterdam, the Netherlands.
Anal Chim Acta. 2024 May 22;1304:342543. doi: 10.1016/j.aca.2024.342543. Epub 2024 Mar 26.
The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this.
In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14-162 Da. 19 of these species have not been previously reported.
The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.
蛋白质的非酶糖基化及其终产物（AGEs）与蛋白质的转化有关，如疾病的发展和生物制药的储存。完整水平上高度糖基化蛋白质的特征描述具有重要意义，因为它允许描述同时发生的蛋白质修饰。然而，糖基化蛋白质的高度异质性使得这一过程具有挑战性，需要新的方法来完成。
在这项研究中，我们研究了两种新的 LC-HRMS 方法，用于在完整蛋白质水平上研究糖基化参考蛋白：低流速亲水相互作用液相色谱（HILIC）和天然尺寸排阻色谱（SEC）。模型蛋白在有利于广泛糖基化和 AGEs 形成的条件下暴露。糖基化后，观察到复杂的 MS 谱，同时信号响应急剧降低，可能是由于蛋白质变性和聚集的形成。当使用 HILIC-MS 时，可以根据还原单糖的数量来分辨蛋白质的糖基化形式。此外，还分离了一些位置糖基化异构体。在非变性条件下使用 SEC-MS 方法可以深入了解糖基化聚集物，但只能根据分子量对糖基化物种进行有限的分离。总的来说，两种方法都观察到了超过 25 种不同类型的物质，分子量相差 14-162 Da。其中 19 种物质以前没有报道过。
所提出的策略具有从天然和变性角度表征高度糖基化完整蛋白质的巨大潜力，为快速临床诊断和研究糖基化相关疾病提供了新的机会。
