Qualitative and quantitative characterization of sialylated N-glycans using three fluorophores, two columns, and two instrumentations
- Authors
- Kim, W.; Kim, J.; You, S.; Do, J.; Jang, Y.; Kim, D.; Lee, J.; Ha, J.; Kim, H.H.
- Issue Date
- Apr-2019
- Publisher
- Academic Press Inc.
- Keywords
- sialylated N-glycan; Fluorescent labeling; HILIC; AEX-HILIC; UPLC; LC-ESI-MS/MS
- Citation
- Analytical Biochemistry, v.571, pp 40 - 48
- Pages
- 9
- Journal Title
- Analytical Biochemistry
- Volume
- 571
- Start Page
- 40
- End Page
- 48
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/18535
- DOI
- 10.1016/j.ab.2019.02.012
- ISSN
- 0003-2697
1096-0309
- Abstract
- Sialylation can influence the stability, half-life, and immunogenicity of glycoproteins, but sialylated N-glycans are known to be difficult to analyze. Human alpha1-acid glycoprotein (AGP) is reported to have glycans that consist of sialylated N-glycans. The N-glycan profiling of AGP is qualitatively and quantitatively investigated here by UPLC and LC-ESI-MS/MS. Three fluorescent tags (AB, AA, and ProA) and two separation columns (HILIC and AEX-HILIC) were adopted to confirm and compare each analytical characteristic. The results of AA were comparable to those of the well-established AB. The qualification of ProA was notable due to its superior fluorescence intensity and ionization efficiency, and ProA showed smaller quantitative or larger-sized fragments in LC-ESI-MS/MS compared to AB and AA. However, the MS quantification of ProA was distorted because the increased sialylation level decreased the LC-ESI-MS/MS ionization efficiency. HILIC had better peak separability, AEX-HILIC had an advantage in UPLC sialylation profiling, and each isomeric glycan could be identified by both columns in LC-ESI-MS/MS. In conclusion, ProA is favored for UPLC and LC-ESI-MS/MS detection but not reliable for MS quantification. This study firstly demonstrates the qualification and quantification of sialylated N-glycans by comparing the commonly used analytical conditions with different fluorescent tags, columns, and instruments. © 2019 Elsevier Inc.
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