Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, Amsterdam 1098 XH, The Netherlands.
Centre for Analytical Sciences Amsterdam, Science Park 904, Amsterdam 1098 XH, The Netherlands.
Anal Chem. 2021 Dec 7;93(48):16000-16007. doi: 10.1021/acs.analchem.1c03473. Epub 2021 Nov 22.
In this study, we optimized a polymerization mixture to synthesize poly(acrylamide--,'-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable ( between 2.5 × 10 and 1.40 × 10 m) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.
在这项研究中，我们优化了聚合混合物，以合成用于完整蛋白质亲水相互作用色谱（HILIC）的聚（丙烯酰胺-甲基丙烯酰胺）整体固定相。进行了热聚合，并研究了改变交联剂用量和致孔剂组成对所得柱分离性能的影响。通过扫描电子显微镜（SEM）检查了结构的均一性和不同的孔隙率。进一步对整体结构进行了表征，发现其为具有渗透性（在 2.5×10 和 1.40×10 m 之间）和极性的固定相，适用于 HILIC。使用含有四种完整蛋白质的样品进行梯度分离，评估了不同柱子的 HILIC 分离性能，性能最佳的固定相在 25 分钟的梯度中表现出 51 的峰容量。与基于硅胶的颗粒酰胺相（2.7μm核壳颗粒）相比，我们比较了基于聚丙烯酰胺的材料。整体固定相没有残留的硅醇基，因此与蛋白质的离子交换相互作用的位点更少。因此，在完整蛋白质的 HILIC 中需要更低浓度的离子对试剂。当使用 0.1%三氟乙酸（TFA）时，两柱的峰容量相似（整体柱和填充柱分别为 30 和 34）。然而，当 TFA 的浓度降低至 0.005%时，整体柱保持相似的分离性能和选择性（峰容量 23），而填充柱的性能则大大降低（峰容量 12），选择性降低，并且无法洗脱所有四种参考蛋白质。最后，使用含有 0.1%甲酸和 0.005%TFA 的流动相，成功地将整体柱上的 HILIC 分离与高分辨率质谱联用。与在 0.1%TFA 下进行的分离相比，蛋白质和糖蛋白的检测灵敏度提高，形成的加合物数量减少。
