Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, Martí i Franquès 1-11, Barcelona 08028, Spain.
J Proteome Res. 2023 Mar 3;22(3):826-836. doi: 10.1021/acs.jproteome.2c00569. Epub 2023 Feb 10.
In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O and N glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N and N glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.
在这项研究中，我们评估了几种色谱吸附剂：多孔石墨碳（PGC）、氨丙基亲水相互作用（aminopropyl-HILIC）和苯硼酸（PBA），用于在线固相萃取毛细管电泳质谱法（SPE-CE-MS）分析糖肽。由于 PBA 吸附剂提供了最有前景的结果，因此开发了一种 PBA-SPE-CE-MS 方法，用于从糖蛋白的酶解物中选择性和灵敏地预浓缩糖肽。选择重组人促红细胞生成素（rhEPO）作为模型糖蛋白，并使用几种蛋白酶进行酶解。靶向 rhEPO 的胰蛋白酶 O 和 N 糖肽以优化方法。在优化条件下，评估了日内精密度、线性、检测限（LOD）和微柱寿命，与之前报道的 TiO-SPE-CE-MS 方法相比，获得了改进的结果，特别是对于 N-糖肽的 LOD（比 CE-MS 低 500 倍，比 TiO-SPE-CE-MS 低 200 倍）。此外，还通过 PBA-SPE-CE-MS 分析了 rhEPO Glu-C 酶解物，以更好地表征 N 和 N 糖肽。最后，该方法用于分析两种 rhEPO 产品（EPOCIM 和 NeuroEPO plus），证明其在生物制药分析中的适用性。所建立的 PBA-SPE-CE-MS 方法提高了现有用于糖肽分析的 CE-MS 方法的灵敏度，并在糖蛋白分析中具有很大的潜力，即使在蛋白质消化物的低浓度下，也可以深入表征蛋白质糖基化位点。
