期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 410, 期 20, 页码 5001-5008出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-018-1150-3
关键词
Glycoproteomics; Glycopeptides; Hemopexin; Hydrophilic interaction liquid chromatography; LC-MS/MS
资金
- Office Of The Director, National Institutes of Health [S10OD023557]
- National Institutes of Health [UO1 CA168926, UO1 CA171146, RO1 CA135069]
- CCSG Grant [P30 CA51008]
The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO (R) penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer armlinked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via alpha 2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.
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