Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
J Chromatogr A. 2021 Jan 11;1636:461793. doi: 10.1016/j.chroma.2020.461793. Epub 2020 Dec 10.
The glycated albumin (G-alb) is a potential marker of hyperglycemia in diabetes and other neurodegenerative disorders in humans. G-alb's presence in the total human serum albumin (tHSA) is an important indicator in the timely diagnosis of disease. To identify G-alb content, it needs to be isolated from non-glycated albumin (NG-alb). Here, we present Capillary electrophoresis (CE) methods with 3-acrylamido phenylboronic acid (3-APBA) as an entrapped ligand in the agarose gel to develop agarose-3-APBA functional capillary and as an affinity ligand added to the buffer without agarose. 3-APBA was selected by computational virtual screening of several phenylboronic acid (PBA) compounds and other ligands to bind G-alb and separate from NG-alb selectively. The agarose-3-APBA functional capillary method involved agarose gel dilution approach coupled with injection pressure to obtain reduced viscosity and sufficient injection volume of protein samples. The method delivered separation in 9.7 min, with a resolution of 3.4, G-alb recovery up to 65%, and took 25 min to complete the entire process. The second method involved 3-APBA as an affinity ligand in the buffer and delivered separation in 4.2 min, with a resolution of 6.4, G-alb recovery up to 102% recovery, with relatively easy procedures. Therefore, it was further applied to determine G-alb content from tHSA in human serum and saliva. The G-alb found content in serum samples was in the range of 21. 1 ± ± 1.4% to 40.5 ± 1.6% out of tHSA and 25.1 ± 1.6% to 33.3 1.4% in saliva. The binding mechanisms were investigated by molecular dockings, which revealed hydrogen bonding, π-π, and van der walls interactions between 3-APBA and G-alb. The affinity was validated by affinity capillary electrophoresis (ACE), which revealed relatively strong interactions between 3-APBA and G-alb with the binding constant (K) of 4.53 × 10M to the 3.41 × 10M of 3-APBA and NG-alb. The affinity of 3-APBA toward G-alb was increased at pH 9.0 of the borax-borate (BB) buffer as background electrolyte (BGE). The limit of detection (LOD) was 10 nM, repeatability (RSD, n = 3) ≤ 1.4%, and recovery rate was 87.8 ± 1.6 to 100 ± 1.4% in serum and 97.3 ± 1.3 to 102.6 ± 1.1% in saliva. The sensitivity and reproducibility of the method met the detection requirements.
糖化白蛋白（G-alb）是糖尿病和其他人类神经退行性疾病中血糖升高的潜在标志物。G-alb 在总人血清白蛋白（tHSA）中的存在是疾病及时诊断的重要指标。为了鉴定 G-alb 含量，需要将其从非糖化白蛋白（NG-alb）中分离出来。在这里，我们提出了毛细管电泳（CE）方法，使用 3-丙烯酰胺基苯硼酸（3-APBA）作为琼脂糖凝胶中的包埋配体，以开发琼脂糖-3-APBA 功能毛细管，并作为亲和配体添加到没有琼脂糖的缓冲液中。3-APBA 通过对几种苯硼酸（PBA）化合物和其他配体的计算虚拟筛选被选中，以与 G-alb 结合并选择性地与 NG-alb 分离。琼脂糖-3-APBA 功能毛细管方法涉及琼脂糖凝胶稀释方法与注射压力相结合，以获得降低的粘度和足够的蛋白质样品注射量。该方法在 9.7 分钟内实现了分离，分辨率为 3.4，G-alb 回收率高达 65%，整个过程耗时 25 分钟。第二种方法涉及 3-APBA 作为缓冲液中的亲和配体，在 4.2 分钟内实现了分离，分辨率为 6.4，G-alb 回收率高达 102%，相对容易进行。因此，它进一步应用于从人血清和唾液中的 tHSA 中测定 G-alb 含量。在血清样品中发现的 G-alb 含量范围为 tHSA 的 21.1%±±1.4%至 40.5%±±1.6%，唾液中的 25.1%±±1.6%至 33.3%1.4%。通过分子对接研究了结合机制，结果表明 3-APBA 与 G-alb 之间存在氢键、π-π 和范德华相互作用。通过亲和毛细管电泳（ACE）验证了亲和力，结果表明 3-APBA 与 G-alb 之间存在相对较强的相互作用，其结合常数（K）为 4.53×10M 至 3.41×10M 的 3-APBA 和 NG-alb。硼酸-硼砂（BB）缓冲液作为背景电解质（BGE）时，3-APBA 对 G-alb 的亲和力在 pH 9.0 时增加。检测限（LOD）为 10 nM，重复性（RSD，n=3）≤1.4%，血清中的回收率为 87.8%±±1.6%至 100%±±1.4%，唾液中的回收率为 97.3%±±1.3%至 102.6%±±1.1%。该方法的灵敏度和重现性满足检测要求。
