Selective Enrichment Of Glycoproteins Research Articles

Comparison of the performance of secretome analysis based on metabolic labeling by three unnatural sugars

分泌蛋白质是调控细胞间信号转导的重要生物大分子。由于分泌蛋白的丰度相比于胞内蛋白以及培养基添加剂更低,因此分泌蛋白的高通量鉴定是目前蛋白质组学界研究的热点和难点。目前,基于生物质谱的分泌蛋白质组学分析一般均需要从无血清的条件培养基中获得分泌蛋白质,再对其进行富集和分析。该流程操作步骤繁琐,易造成分泌蛋白质的损失和降解。本工作采用基于生物正交化学生物学技术实现对分泌蛋白质的高选择性标记和高效富集。通过结合点击化学技术,综合评估了分泌蛋白质分析中用于代谢标记的不同糖类似物。采用3种最常用的商品化糖类似物,N-叠氮乙酰甘露糖胺(ManNAz)、N-叠氮乙酰半乳糖胺(GalNAz)和N-叠氮乙酰葡萄糖胺(GlcNAz)分别对HeLa细胞进行代谢标记,之后通过炔基生物素探针对条件培养基中的分泌蛋白进行富集,结合质谱分析来对比3种糖类似物对分泌蛋白的标记效率。最后通过无标定量蛋白质组学分析,系统评估了3种糖类似物用于分泌蛋白质组分析的性能。结果表明,基于ManNAz的分泌蛋白标记方法鉴定到了282个分泌蛋白、224个细胞质膜蛋白以及846个N-糖基化位点;对分泌蛋白的富集效率分别较GalNAz和GlcNAz提高了130%和67.2%;对细胞质膜蛋白的富集效率较GalNAz和GlcNAz分别提高了273.3%和148.7%,体现出了明显的优势。本研究的实验结果为分泌蛋白高选择性富集和系统分析提供了有益的对比分析和新技术策略。

Boronic acid-functionalized spherical polymer brushes for efficient and selective enrichment of glycoproteins.

Glycoproteins are related to many biological activities and diseases, and thereby their efficient capture and enrichment for diagnostics and proteomics have emerged to exhibit great significance. However, the lack of materials with high binding capacity and selectivity is still a big obstacle for further application. Herein, we reported a facile and eco-friendly approach to fabricate spherical polymer brushes with multiple boronic acid groups. Specifically, the whole process can be divided into three steps, the polystyrene (PS) core was obtained by traditional emulsion polymerization, followed by immobilization of a home-made photoinitiator. Subsequently, boronic acid-functionalized polymer chains (PBA) were chemically grafted via photo-emulsion polymerization, leading to spherical polymer brushes (PS-PBA) with boronate affinity. The particle size, morphology, and composition of as-prepared spherical polymer brushes were systematically characterized. The characteristics of glycoproteins binding to the spherical polymer brushes under different conditions, including pH values and ionic strength, were also investigated. PS-PBA brushes possess fast binding speed (30 min) and high binding capacity for glycoprotein ovalbumin (OVA) (377.0 mg g-1) under physiological pH conditions at 25 °C, because the low steric hindrance of flexible polymeric PBA chains facilitates the interaction between boronic acid groups and glycoproteins. Moreover, the binding capacity of PS-PBA brushes for glycoprotein OVA was ∼6.7 times higher than that for non-glycoprotein bovine serum albumin (BSA), indicating the excellent selective adsorption. This study provided a facile and efficient approach for the fabrication of boronic acid-functionalized materials that will be useful in the enrichment and separation of glycoproteins for the diagnosis of diseases.

Preparation of Boronic Acid-Functionalized Silica Nanocomposites for Selective Enrichment of Glycoproteins.

A facile method was developed for synthesis of boronic acid-functionalized silica nanocomposites (SiO2 -BA) by 'thiol-ene' click reaction, where silica nanoparticles were synthesized by using tetraethoxysilane (TEOS) and γ-mercaptopropyl trimethoxysilane (γ-MPTS) as precursors. The morphology and structure properties of the resultant SiO2 -BA were characterized by transmission electronic microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and Brunner-Emmet-Teller measurements (BET). The adsorption behavior of the SiO2 -BA for glycoproteins was evaluated. Under the optimized conditions, the SiO2 -BA exhibited higher adsorption capacity towards glycoproteins (ovalbumin, OVA, 7.64 μmol/g) than non-glycoproteins (bovine serum albumin, BSA, 0.83 μmol/g). In addition, the practicality of the SiO2 -BA was further assessed by selective enrichment of glycoproteins from egg white samples.

Method development of glycoprotein biomarkers for cancers.

Method development of glycoprotein biomarkers for cancers.

Recent advances in the application of core–shell structured magnetic materials for the separation and enrichment of proteins and peptides

Many endogenous proteins/peptides and proteins/peptides with post-translational modifications (PTMs) are presented at extremely low abundance, and they usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis. Therefore, the separation and enrichment of proteins/peptides from complex mixtures is of great importance to the successful identification of them. Core–shell structured magnetic microspheres have been widely used in the enrichment and isolation of proteins/peptides, thanks to unique properties such as strong magnetic responsiveness, outstanding binding capacity, excellent biocompatibility, robust mechanical strength and admirable recoverability. The aim of this review is to update the advances in the application of core–shell structured magnetic materials for proteomics analysis, including the separation and enrichment of low-concentration proteins/peptides, the selective enrichment of phosphoproteins and the selective enrichment of glycoproteins, and to compare the enrichment performance of magnetic microspheres with different kinds of functionalization.