Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, P.R. China.
College of Chemistry and Molecular Sciences, Wuhan University , Wuhan 430072, P.R. China.
Anal Chem. 2017 Sep 19;89(18):9712-9721. doi: 10.1021/acs.analchem.7b01283. Epub 2017 Aug 31.
Protein glycosylation is an important post-translational modification that plays a crucial role in many biological processes. Because of the low abundance of glycoproteins and high complexity of clinical samples, the development of methods to selectively capture glycoproteins/glycopeptides is crucial to glycoproteomics study. In this work, a kind of highly cross-linked chitosan microspheres (CSMs) was prepared using epichlorhydrine as a cross-linker from chitosan solution in an alkaline/urea aqueous system. The results showed that CSMs had high amino groups content, large surface area, mesoporous structure, good acidic resistance, and high strength by various tests. On the basis of hydrophilic interaction between the polar groups (amino groups and hydroxyl groups) on CSMs and glycan moieties on glycopeptides, the prepared CSMs were applied to specific capture of N-glycopeptides from standard protein digests and complex biological samples (body fluids and tissues). The CSMs exhibited high selectivity (HRP/BSA = 1:100), good sensitivity (4.5 × 10 M of HRP), good recovery yield (74.9-106.4%), and high binding capacity (100 mg g) in glycopeptides enrichment. Because of the excellent performance in glycopeptides enrichment, CSMs were applied to selectively enrich N-glycopeptides from tryptic digests of human serum and rat brain followed by nanoLC-MS/MS analysis. We identified 194 and 947 unique N-glycosylation sites from 2 μL of human serum and 0.1 mg of rat brain, respectively. Additionally, the extraction time of our method was much shorter than the previously reported methods. Therefore, the fabricated CSMs with desirable properties will find broad application in large-scale and in-depth N-glycoproteome analysis.
蛋白质糖基化是一种重要的翻译后修饰，在许多生物过程中起着至关重要的作用。由于糖蛋白的丰度低和临床样本的复杂性高，因此开发选择性捕获糖蛋白/糖肽的方法对于糖蛋白质组学研究至关重要。在这项工作中，使用环氧氯丙烷作为交联剂，从碱性/尿素水溶液中的壳聚糖溶液中制备了一种高度交联的壳聚糖微球（CSM）。通过各种测试表明，CSM 具有高氨基含量、大表面积、中孔结构、良好的耐酸性和高强度。基于 CSM 上的极性基团（氨基和羟基）与糖肽上的糖基部分之间的亲水性相互作用，制备的 CSM 被应用于从标准蛋白质消化物和复杂生物样品（体液和组织）中特异性捕获 N-糖肽。CSM 表现出高选择性（HRP/BSA = 1:100）、良好的灵敏度（HRP 的 4.5×10-9 M）、良好的回收率（74.9-106.4%）和高结合能力（100mg g-1）在糖肽富集方面。由于在糖肽富集方面的优异性能，CSM 被应用于选择性富集人血清和大鼠脑的胰蛋白酶消化物中的 N-糖肽，然后进行纳升液相色谱-串联质谱（nanoLC-MS/MS）分析。我们从 2 μL 人血清和 0.1mg 大鼠脑中分别鉴定出 194 个和 947 个独特的 N-糖基化位点。此外，我们方法的提取时间比以前报道的方法短得多。因此，具有理想性能的制备的 CSM 将在大规模和深入的 N-糖蛋白质组学分析中得到广泛应用。
