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Qualitative and quantitative characterization of sialylated N-glycans using three fluorophores, two columns, and two instrumentations

Kim, Wooseok, Kim, Jihye, You, Seungkwan, Do, Jonghye, Jang, Yeonjoo, Kim, Donghwi, Lee, Junmyoung, Ha, Jongkwan, Kim, Ha Hyung
Analytical biochemistry 2019
fluorescence, fluorescent dyes, glycoproteins, half life, humans, hydrophilic interaction chromatography, immunogenicity, ionization, polysaccharides, tandem mass spectrometry
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.