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Comparison of reverse phase chromatography- and hydrophilic interaction chromatography-tandem mass spectrometry in the study and characterization of O-sialoglycopeptides from hydrolyzed caseinomacropeptide

AutorHernández-Hernández, Oswaldo ; Lebrón-Aguilar, Rosa ; Quintanilla-López, Jesús Eduardo ; Sanz, M. Luz ; Moreno, F. Javier
Fecha de publicación2010
CitaciónHTC-11 (2010)
HTSP (2010)
ResumenProteins can be found in nature glycosylated with different carbohydrates by both N- and O- linkages. O-linked glycosylation consists of attaching the glycans to the hydroxyl oxygen of serine and threonine residues in sequence regions of high hydroxyamino acid density without a unique sequence motif required for this attachment. Considering the heterogeneity of both peptide and oligosaccharide structures for O-glycoproteins, their analysis is not trivial and advanced analytical techniques are required. Different HPLC separation modes (i.e. size exclusion, anion exchange, reverse phase (RP), amine-bonded silica phases, etc) are commonly used to analyse glycoproteins. RP-HPLC is the most extended among them, but in the last decade, hydrophilic interaction liquid chromatography (HILIC) is gaining a great importance in the analysis of glycopeptides. In recent times, N-glycopeptides have been widely analysed by HPLC tandem mass spectrometry (MS) but little information is found about O-glycopeptides. Therefore, the aim of this work was to develop a HILIC-MS method to characterize O-sialoglycopeptides in a tryptic/chymotryptic hydrolyzate of bovine caseinomacropeptide and to compare its efficiency with the RP-HPLC method. Behaviour of non-glycosylated peptides was also studied. Peptides were separated on a zwitterionic HILIC column using an elution gradient of acetonitrile and water with 0.005% formic acid. For RP-HPLC method a Phenomenex Jupiter Proteo column with a gradient of acetonitrile and water with 0.1% of formic acid was used. Detection for both chromatographic modes was carried out on an ion trap mass spectrometer in positive mode. Different separation behaviour for both non-glycosylated peptides and O-glycopeptides by RP-HPLC and HILIC was observed. Non-glycosylated peptides were more retained than those glycosylated by RP-HPLC; however, although more complex chromatograms were obtained by HILIC, the separation achieved by this technique was better for the O-sialoglycopeptides. Therefore, the optimized method made the detection of peptides by MSn easier. LC-MS2 analyses of O-glycopeptides mainly resulted in glycosydic bond fragmentation with the corresponding ion of the intact peptidic chain and the respective losses of carbohydrates units. To confirm the amino acidic sequences of glycopeptides, MS3 spectra were succesfully performed from the ion corresponding to the intact peptidic chain. This method allowed the identification and characterization of ~30 O-glycopeptides. In conclusion, HILIC-MS has shown to be a powerful technique for the identification of O-glycopeptides, allowing the detection of a higher number of compounds than RP-HPLC without any previous derivatization treatment.
DescripciónResumen del trabajo presentado al Eleventh International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers and International Symposium on Hyphenated Techniques for Sample Preparation celebrados en Brujas (Bélgica) del 26 al 29 de enero de 2010.
Aparece en las colecciones: (CIAL) Comunicaciones congresos
(IQOG) Comunicaciones congresos
(IQFR) Comunicaciones congresos
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