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Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides
P. Kozlik, M. Sanda, R. Goldman,
Language English Country Netherlands
Document type Journal Article
- MeSH
- Chemistry Techniques, Analytical methods standards MeSH
- Chromatography, Liquid * MeSH
- Chromatography, Reverse-Phase * MeSH
- Glycopeptides analysis MeSH
- Hemopexin analysis MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Polysaccharides chemistry MeSH
- Tandem Mass Spectrometry MeSH
- Publication type
- Journal Article MeSH
Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.
References provided by Crossref.org
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- $a Kozlik, Petr $u Department of Oncology, Lombardi Comprehensive Cancer Center PSB GF9, Georgetown University, 3800 Reservoir Road NW, Washington, DC 20057, United States; Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague 2, Czech Republic.
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- $a Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.
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- $a Goldman, Radoslav $u Department of Oncology, Lombardi Comprehensive Cancer Center PSB GF9, Georgetown University, 3800 Reservoir Road NW, Washington, DC 20057, United States; Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, 3800 Reservoir Road NW, Washington, DC 20057, United States. Electronic address: rg26@georgetown.edu.
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