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, 08028, Barcelona, Spain.
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, 08028, Barcelona, Spain.
Talanta. 2020 Mar 1;209:120563. doi: 10.1016/j.talanta.2019.120563. Epub 2019 Nov 15.
In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L and 10-50 mg L for O glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.
本研究描述了一种在线二氧化钛固相萃取毛细管电泳-质谱联用（TiO-SPE-CE-MS）方法，用于分析重组人红细胞生成素（rhEPO）胰蛋白酶消化物中获得的糖肽糖型。由于 rhEPO 的 O-糖肽在生物制药质量控制和兴奋剂分析中的重要性，因此将其用于优化方法。研究了影响 O 糖型选择性保留和洗脱、峰效率和电泳分离的几个方面，以最大限度地提高检测灵敏度，同时最小化肽的非特异性保留。在优化的条件下，微柱寿命约为 10 次分析，重复性可接受（峰迁移时间和峰面积的 %RSD 值分别为 9-11%和 6-11%）。该方法在线性范围为 0.5-50mg/L 和 10-50mg/L 之间，对于含有 NeuAc 和 NeuGc 的 O 糖型分别为 50mg/L，检测限（LOD）比 CE-MS 低 100 倍。虽然该方法是针对 O-糖肽进行优化的，但也成功地用于浓缩 N-糖肽，而不会影响具有不同唾液酸数量的糖肽糖型之间的分离。还分析了其他糖蛋白（即人载脂蛋白 CIII（APO-C3）和牛α-1-酸性糖蛋白（bAGP））的胰蛋白酶消化物，证明了该方法适用于具有不同聚糖组成和性质的糖肽。
