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Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode
K. Molnarova, A. Duris, T. Jecmen, P. Kozlik
Language English Country Germany
Document type Comparative Study, Journal Article
Grant support
19-18005Y
Akademie Věd České Republiky
SVV260560
charles university
LTC20078
Ministry of Education, Youth and Sports of the Czech Republic
NLK
ProQuest Central
from 2011-01-01 to 1 year ago
Medline Complete (EBSCOhost)
from 2003-01-01 to 1 year ago
Health & Medicine (ProQuest)
from 2011-01-01 to 1 year ago
- MeSH
- Chromatography, Ion Exchange methods MeSH
- Chromatography, Reverse-Phase methods MeSH
- Glycopeptides isolation & purification MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Immunoglobulin G isolation & purification MeSH
- Isomerism MeSH
- Humans MeSH
- Proteomics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.
References provided by Crossref.org
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