Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS.
Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS.
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DOI：
10.1039/d2an00010e
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发表时间：
2022-05-17
期刊：
影响因子：
4.2
通讯作者：
Mechref, Yehia
中科院分区：
文献类型：
--
作者：
Wang, Junyao;Dong, Xue;Yu, Aiying;Huang, Yifan;Peng, Wenjing;Mechref, Yehia
糖基化是影响蛋白质性质和功能的重要生物学过程。已显示聚糖异构体参与多种疾病进展。然而，由于聚糖异构体结构的微观异质性，聚糖异构体的分离和鉴定多年来一直是一个挑战。因此，在过去的几十年里，人们研究了有效和稳定的技术来改善聚糖的异构体分离。反相高效液相色谱法因其在保留时间和分离分辨率方面具有良好的重现性和可靠性而被广泛应用于生物分子分析。然而，到目前为止，还没有研究使用该技术实现聚糖异构体的高分辨率。在这项研究中，我们专注于使用500 mm反相LC柱进一步提高全甲基化聚糖的异构体分离。为了实现全甲基化聚糖的更好分离度，使用聚糖标准品优化了不同的LC条件，包括核心和支链岩藻糖基化N-聚糖异构体和唾液酸连接的异构体，这两种异构体均成功分离。然后，将优化的分离策略应用于牛胎球蛋白、核糖核酸酶B和κ-酪蛋白等模型糖蛋白的N-和O-聚糖异构体的分离。在多种唾液酸化键合异构体上观察到基线分离。但对高甘露糖异构体的分离性能有待进一步提高。还通过对多个聚糖的保留时间进行运行间、日间和月间比较，检测了该长C18色谱柱的重现性和稳定性，发现%RSD小于0.92%。最后，我们应用这种方法来分离聚糖异构体来自复杂的生物样品，包括血清和细胞系，在几个异构体结构上实现基线分离。与PGC和MGC色谱柱的分离效率相比，RPLC C18色谱柱的分离度较低，但重现性更稳定，这使其成为聚糖异构体分离的良好补充选择。采用LC-MS/MS和500 mm反相C18色谱柱分离来自模型糖蛋白、人血清和癌细胞系的含唾液酸键异构体和位置岩藻糖基化异构体的聚糖。
Glycosylation is known as a critical biological process that can largely affect the properties and the functions of proteins. Glycan isomers have been shown to be involved in a variety of disease progressions. However, the separation and identification of glycan isomers has been a challenge for years due to the microheterogeneity of glycan isomeric structures. Therefore, effective and stable techniques have been investigated over the last few decades to improve isomeric separations of glycans. RPLC has been widely used in biomolecule analysis because of its extraordinary reproducibility and reliability in retention time and separation resolution. However, so far, no studies have achieved high resolution of glycan isomers using this technique. In this study, we focused on further boosting the isomeric separation of permethylated glycans using a 500 mm reversed-phase LC column. To achieve better resolutions on permethylated glycans, different LC conditions were optimized using glycan standards, including core- and branch-fucosylated N-glycan isomers and sialic acid linked isomers, which were both successfully separated. Then, the optimal separation strategy was applied to achieve separations of N- and O- glycan isomers derived from model glycoproteins, including bovine fetuin, ribonuclease B and κ- casein. Baseline separations were observed on multiple sialylated linkage isomers. However, the separation performance of high-mannose isomers needs further improvement. The reproducibility and stability of this long C18 column was also tested by doing run-to-run, day-to-day and month-to-month comparisons of retention times on multiple glycans and the %RSD was found less than 0.92%. Finally, we applied this approach to separate glycan isomers derived from complex biological samples, including blood serum and cell lines, where baseline separations were attained on several isomeric structures. Compared to the separation efficiency of PGC and MGC columns, the RPLC C18 column provides lower resolution but more robust reproducibility, which makes it a good complementary alternative for isomeric separations of glycans. Separation of glycans with sialic acid linkage isomers and positional fucosylated isomers derived from model glycoproteins, human serum and cancer cell lines using LC-MS/MS with a 500 mm reversed-phase C18 column.
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影响因子：
7.4
作者：
Goetz, John A.;Novotny, Milos V.;Mechref, Yehia
通讯作者：
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影响因子：
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影响因子：
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通讯作者：
Roussel, P
影响因子：
4.3
作者：
LAINE, RA
通讯作者：
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