Isomeric separation of native N-glycans using nano zwitterionic- hydrophilic interaction liquid chromatography column

Changes in the expression of glycan isomers have been implicated in the development and progression of several diseases. However, the analysis of structurally diverse isomeric N-glycans by LC -MS/MS is still a major analytical challenge, particularly due to their large number of possible isomeric conformations. Common approaches derivatized the N-glycans to increase their hydrophobicity and to gain better detec-tion in the MS system. Unfortunately, glycan derivatization is time-consuming and, in many cases, adds complexity because of the multiple reaction and cleaning steps, incomplete chemical labeling, possible degradation, and unwanted side reactions. Thus, analysis of native glycans, especially for samples with low abundance by LC -MS/MS, is desirable. Normal phase chromatography, which employs HILIC station-ary phase, has been commonly employed for the identification and separation of labeled glycans. In this study, we focused on achieving efficient isomeric separation of native N-glycans using a nano ZIC-HILIC column commonly employed to separate labeled glycans and glycopeptides. Underivatized sialylated and oligomannose N-glycans derived from bovine fetuin and Ribonuclease B were initially utilized to optimize chromatographic conditions, including column temperature, pH of mobile phases, and gradient elution time. The optimized condition was then applied for the isomeric separation of native N-glycans derived from alpha-1 acid glycoprotein, as well as from biological samples. Finally, we confirmed the stability and reproducibility of the ZIC-HILIC column by performing run-to-run comparisons of the full width at half height (FWHM) and retention time on different N-glycans. The variability in FWHM was less than 0.5 min, while that of retention time was less than 1.0 min with %RSD less than 1.0%.& COPY; 2023 Published by Elsevier B.V.

Automated summary

Changes in glycan expression play a role in the development of diseases. Analyzing isomeric N-glycans using LC-MS/MS is challenging due to the large number of possible conformations. Derivatization of glycans is commonly used but time-consuming. This study focuses on efficient isomeric separation of native N-glycans using a nano ZIC-HILIC column. The stability and reproducibility of the column were confirmed.