Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France.
Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France; Sorbonne Université, Paris, France.
J Pharm Biomed Anal. 2020 Jan 30;178:112921. doi: 10.1016/j.jpba.2019.112921. Epub 2019 Oct 13.
Glycosylation is one of the most common post-translational modifications of proteins that affects their biological activity, solubility, and half-life. Therefore, its characterization is of great interest in proteomic, particularly from a diagnostic and therapeutic point of view. However, the number and type of glycosylation sites, the degree of site occupancy and the different possible structures of glycans can lead to a very large number of isoforms for a given protein, called glycoforms. The identification of these glycoforms constitutes an important analytical challenge. Indeed, to attempt to characterize all of them, it is necessary to develop efficient separation methods associated with a sensitive and informative detection mode, such as mass spectrometry (MS). Most analytical methods are based on bottom-up proteomics, which consists in the analysis of the protein at the glycopeptides level after its digestion. Even if this approach provides essential information, including the localization and composition of glycans on the protein, it is also characterized by a loss of information on macro-heterogeneity, i.e. the nature of the glycans present on a given glycoform. The analysis of glycoforms at the intact level can overcome this disadvantage. The aim of this review is to detail the state-of-the art of separation methods that can be easily hyphenated with MS for the characterization of protein glycosylation at the intact level. The different electrophoretic and chromatographic approaches are discussed in detail. The miniaturization of these separation methods is also discussed with their potential applications. While the first studies focused on the development and optimization of the separation step to achieve high resolution between isoforms, the recent ones are much more application-oriented, such as clinical diagnosis, quality control, and glycoprotein monitoring in formulations or biological samples.
糖基化是蛋白质最常见的翻译后修饰之一，它影响蛋白质的生物活性、溶解度和半衰期。因此，从蛋白质组学的角度来看，特别是从诊断和治疗的角度来看，对其进行特征描述具有重要意义。然而，糖基化位点的数量和类型、位点占有率的程度以及聚糖的不同可能结构会导致给定蛋白质产生非常多的异构体，称为糖型。这些糖型的鉴定构成了一个重要的分析挑战。事实上，为了尝试对所有糖型进行特征描述，有必要开发高效的分离方法，并结合灵敏和信息丰富的检测模式，如质谱（MS）。大多数分析方法基于自上而下的蛋白质组学，即对蛋白质进行消化后在糖肽水平上进行分析。尽管这种方法提供了包括糖基在蛋白质上的定位和组成在内的重要信息，但它也存在一个缺点，即无法获取关于宏观异质性的信息，即给定糖型上存在的聚糖的性质。对完整糖型的分析可以克服这一缺点。本文的目的是详细介绍可以与 MS 轻松联用的分离方法，用于完整水平上的蛋白质糖基化特征描述。详细讨论了不同的电泳和色谱方法。还讨论了这些分离方法的微型化及其潜在应用。虽然早期的研究集中在开发和优化分离步骤以实现异构体之间的高分辨率，但最近的研究更侧重于应用，例如临床诊断、质量控制以及制剂或生物样品中糖蛋白的监测。
