ReviewApplication of magnetic solid phase extraction in separation and enrichment of glycoproteins and glycopeptides
Graphical abstract
This review mainly focus on the recent advances in the application of magnetic solid phase extraction (MSPE) technology and magnetic adsorbents for the separation and enrichment of glycoproteins and glycopeptides in human body fluids.
Introduction
Protein glycosylation plays an important role in living organisms, which not only participates in many biological processes such as protein translation regulation, protein degradation, immune protection and signal transduction regulation, but also has an important impact on protein characteristics such as structure, solubility, charge or sensitivity to proteolysis [[1], [2], [3]]. Human body fluids are one of the main sources for the discovery of related disease biomarkers, and often used as clinical biological specimens [4,5]. Changes in content of glycoproteins and glycopeptides in body fluids usually reflect the physical status of human body [6]. Therefore, the quantitative and qualitative analysis of glycoproteins and glycopeptides in human body fluids are crucial for the screening and discovery of clinical disease biomarkers. Mass spectrometry (MS) analysis is a powerful and high-throughput method that plays a central role in glycomics and glycoproteomics [7,8]. Nevertheless, systematic and comprehensive analysis of protein glycosylation is still challenging [9,10]. For one thing, the high abundance of non-glycoproteins and non-glycopeptides seriously hinders the detection of low-abundance glycoproteins and glycopeptides in complex biological samples. For another, the heterogeneity of glycans further reduces the relative content of each glycan form. In addition, the analysis of glycosylation sites and glycan components requires complex bioinformatics software to explain massive mass spectrometry data. Hence, developing an effective pre-concentration method is very important to overcome these obstacles and enables the comprehensive analysis of glycoproteins and glycopeptides with MS.
Up to now, the specific enrichment methods for glycoproteins and glycopeptides mainly include hydrophilic interaction liquid chromatography (HILIC) [[11], [12], [13]], boronate affinity chromatography [14,15], hydrazine chemical method [16,17], and lectin affinity chromatography [18]. The enrichment mechanism of HILIC is primarily the hydrophilic distribution of the compound between the organic-rich mobile phase and the water-rich layer formed on the adjacent surface of the HILIC stationary phase [19]. What is more, hydrogen bonding and electrostatic interactions also participate in the retention of HILIC [20]. The mechanism of boronate affinity chromatography can be explained by the reversible formation/dissociation of covalent complexes between boric acid groups and compounds containing cis-diol groups in alkaline/acidic aqueous solutions [21]. In the hydrazide chemistry method, cis-diol groups of glycoproteins and glycopeptides are usually oxidized into aldehydes by periodate, followed by the covalent bonding with hydrazide [22]. Lectin affinity chromatography is a highly specific method for capturing glycoproteins and glycopeptides. Its enrichment mechanism depends on the biological specific recognition between lectins and glycans. Usually, a lectin can only recognize a subset of glycoproteins [23]. In recent years, enrichment materials based on the above enrichment strategies have been widely developed to meet the requirements of low-abundance glycoproteins and glycopeptides enrichment in complex samples.
Magnetic solid phase extraction (MSPE) is widely employed in glycoproteomics because of its simple operation, short adsorption and desorption time, low cost and ecological friendliness [24]. As the core of MSPE technology, magnetic adsorbents possess the ability of rapid separation of target analytes, which benefiting from the excellent magnetic responsiveness of Fe3O4 nanoparticles. However, the practical application of Fe3O4 is restricted due to its high surface activity, easy agglomeration and unstable long-term storage. Therefore, the reasonable functionalization of Fe3O4 is crucial, which can not only improve surface properties and stability, but also broaden the scope of its application [25]. So far, some reviews have been reported for the applications of magnetic materials in the enrichment of glycoproteins and glycopeptides. In 2014, Deng's group [26] reviewed the synthesis of core-shell magnetic microspheres and their applications in protein sample preparation, including the separation and enrichment of phosphoproteins, glycoproteins and other low-abundance proteins/peptides. In 2020, Cao’s group [27] described the preparation and functionalization of boronate affinity-based magnetic composites, and summarized the applications of boronate affinity magnetic separation technology in affinity separation, proteomics, detection methods and enzyme immobilization.
In this review, we focus on the application of MSPE technology and magnetic adsorbents for the separation and enrichment of glycoproteins and glycopeptides in human body fluids (Fig. 1). Firstly, the principle and development of MSPE technology are briefly outlined. Secondly, magnetic adsorbents of MSPE are classified based on different functional materials and discussed one by one. Thirdly, the application of MSPE technology for glycoproteins and glycopeptides enrichment in different human body fluids is summarized and commented. Finally, the future trends and application prospects of MSPE technology in glycoproteomics are described.
Section snippets
Overview of MSPE technology
The concept of MSPE was firstly proposed by Šafaříková and Šafařík in 1999 as a new sample pretreatment technology based on solid phase extraction (SPE) [28]. MSPE technology combines magnetic separation and SPE, utilizing magnetic materials as adsorbents to separate and enrich target analytes. It overcomes the defects of traditional SPE such as easy blockage, time consumption and low extraction efficiency, and realizes the rapid separation and enrichment of targets in complex matrix [29]. With
Categories of magnetic adsorbents
Magnetic adsorbents have been considered as the key to realize rapid separation and efficient enrichment. Typically, magnetic adsorbents consist of magnetic particles and their surface-modified functional materials. Magnetic particles realize the rapid separation of the target analytes from the sample solution, while the types of functional materials affect the sensitivity and selectivity of separation and enrichment [31]. Among these magnetic particles such as metals (Fe, Co, Ni), alloys
Application of MSPE in human body fluids
Studies have shown that 70% of human proteins contain one or more sugar chains, and 1% of human genome participates in the synthesis and modification of sugar chains [66,67]. The changes in the kinds and concentration of glycoproteins in human body fluids can not only reflect the glycosylation level of proteins, but also make contributions to the search and discovery of disease biomarkers [68]. Therefore, the qualitative and quantitative analysis of glycoproteins and glycopeptides in human body
Conclusion and perspective
In summary, glycoproteins and glycopeptides are vulnerable to interference and ion inhibition. Therefore, effective separation and enrichment are required prior to MS detection. In the past decade, novel strategies used for the separation and enrichment of glycoproteins and glycopeptides have sprung up. With the aid of these strategies, improved enrichment efficiency, selectivity and sensitivity of glycoproteins and glycopeptides have been realized, which are beneficial to the analysis and
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the Natural Science Foundation of Jilin Provincial Science & Technology Department (No. 20190201079JC).
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