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Development of a new method using HILIC-tandem mass spectrometry for the characterization of O-sialoglycopeptides from proteolytically digested caseinomacropeptide

AuthorsHernández-Hernández, Oswaldo ; Lebrón-Aguilar, Rosa ; Quintanilla-López, Jesús Eduardo ; Sanz, M. Luz ; Moreno, F. Javier
Ion trap
Issue DateOct-2010
CitationProteomics 10(20): 3699-3711 (2010)
AbstractThis work addresses the optimization of HILIC (hydrophilic interaction liquid chromatography)-ESI-MSn conditions for the comprehensive characterization of O-glycopeptides from proteolytically digested caseinomacropeptide. O-Glycopeptides were satisfactorily analysed on a zwitterionic HILIC column based on their glycan structure and amino acid sequence. The contribution of ionic interactions to the retention of charged glycopeptides was found to be substantial. Thus, O-glycopeptides carrying neutral glycans were more retained than O-sialoglycopeptides because negatively charged sialic acid residues were electrostatically repelled by the stationary phase. In addition, glycopeptides differing only in the position of the linkage of the sialic acid moiety could be separated. The same chromatographic behaviour was observed for model systems constituted by a synthetic tetrapeptide covalently conjugated to neutral and sialylated carbohydrates. Subsequent detection of caseinomacropeptide O-glycopeptides was carried out on an electrospray ion trap tandem mass spectrometer at both positive and negative ionization modes. MS2 fragmentation at positive ionization mode was valid for determining the glycan structure as the resulting main fragments corresponded to Yn-type ions derived from sequential glycosidic bond fragmentation, whilst the fragmentation of the peptide structure was preferably obtained through the formation of bn-type ions at the MS3 stage, allowing the complete structure elucidation of the peptidic chain. Overall, the developed method allowed the identification and characterization of 41 O-glycopeptides covering all the known glycosylation sites without any previous enrichment step. These results point out that HILIC coupled to multistage MS procedures can be a powerful technique for future glycoproteomic applications.
Description13 páginas, 10 figuras, 2 tablas.-- El pdf del artículo es la versión post-print.
Publisher version (URL)http://dx.doi.org/10.1002/pmic.201000156
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