Gautam Sakshi, Banazadeh Alireza, Cho Byeong Gwan, Goli Mona, Zhong Jieqiang, Mechref Yehia
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, United States.
Anal Chem. 2021 Mar 30;93(12):5061-5070. doi: 10.1021/acs.analchem.0c04395. Epub 2021 Mar 15.
Post-translational modifications are vital aspects of functional proteins. Therefore, it is critical to understand their roles in biological processes. Glycosylation is particularly challenging to study among these modifications due to the heterogeneity displayed by the glycans in terms of their isomers. Thus, researchers continue to strive for the development of efficient liquid chromatography techniques for isomeric separation of glycans. Porous graphitized carbon (PGC) nano column has been one of the most widely used columns for this purpose, but poor stability and lack of reproducibility led to its discontinuation. In our endeavor to find an alternative stationary phase for isomeric glycan separation, we tested the mesoporous graphitized carbon (MGC) material. Unprecedentedly, satisfactory results were obtained with a column only 1 cm long, which was tested on permethylated -glycans derived from model glycoproteins as well as biological samples. The column was found to be reproducible across months as well as across different column preparations. Additionally, to decrease the dead volume and attain a better resolution, MGC was utilized to pack a 1 cm length of a pulled capillary nanospray emitter and again demonstrated efficient isomeric separation. Thus, MGC proved to be a suitable stationary phase to obtain efficient isomeric separation of permethylated -glycans with 1 cm-long packing length, in both capillary columns and packed nanospray emitters.
翻译后修饰是功能蛋白的重要方面。因此，了解它们在生物过程中的作用至关重要。在这些修饰中，糖基化由于聚糖异构体表现出的异质性而特别具有研究挑战性。因此，研究人员继续致力于开发用于聚糖异构体分离的高效液相色谱技术。多孔石墨化碳（PGC）纳米柱一直是为此目的使用最广泛的柱之一，但稳定性差和缺乏重现性导致其停用。在我们寻找用于异构体聚糖分离的替代固定相的努力中，我们测试了介孔石墨化碳（MGC）材料。前所未有的是，使用仅1厘米长的柱获得了令人满意的结果，该柱在源自模型糖蛋白以及生物样品的全甲基化聚糖上进行了测试。发现该柱在数月以及不同的柱制备之间具有重现性。此外，为了减少死体积并获得更好的分辨率，利用MGC填充1厘米长的拉制毛细管纳米喷雾发射器，并再次证明了有效的异构体分离。因此，MGC被证明是一种合适的固定相，可在毛细管柱和填充纳米喷雾发射器中以1厘米的填充长度实现全甲基化聚糖的高效异构体分离。
