Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic.
Department of Biochemistry, Faculty of Science, Charles University, 128 00, Prague 2, Czech Republic.
Anal Bioanal Chem. 2021 Jul;413(16):4321-4328. doi: 10.1007/s00216-021-03388-3. Epub 2021 May 17.
Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.
糖蛋白质组学是蛋白质组学中具有挑战性的分支，因为蛋白质糖基化具有微观和宏观的不均一性。亲水作用色谱（HILIC）是反相色谱法的有利替代方法，可在通过质谱法鉴定完整糖肽之前对其进行分离。如今，有几种在所用化学性质上有所不同的 HILIC 柱在商业上可利用。但是，缺乏评估其性能的比较研究，因此无法为不同糖蛋白质组学应用选择合适的固定相提供指导。在这里，我们将最近由先进色谱技术公司（ACE）开发的三种 HILIC 柱（未功能化的 HILIC-A、多羟基功能化的 HILIC-N 和氨丙基功能化的 HILIC-B）与 C18 反相柱进行了比较，用于分离人免疫球蛋白 G 糖肽。HILIC-A 和 HILIC-B 表现出混合模式分离，结合了亲水和离子交换相互作用以保留分析物。可以预期，反相模式成功地分离了免疫球蛋白 G1 和免疫球蛋白 G2 糖肽的簇，它们在氨基酸序列上有所不同，但不能充分分离同一肽的不同糖型。所有 ACE HILIC 柱对不同糖型的分离能力都更高，我们表明，每根柱都能更有效地分离出不同的糖肽群，比其他柱更有效。此外，HILIC-A 和 HILIC-N 柱还分离了免疫球蛋白的等电 A2G1F1 糖肽，因此显示出阐明同型糖型结构的潜力。此外，还根据确定的色谱参数讨论了 HILIC 柱的可能保留机制。
