Identification of Sialylated Oligosaccharides Derived from Ovine and Caprine Caseinomacropeptide by Graphitized Carbon L
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Identification of Sialylated Oligosaccharides Derived from Ovine and Caprine Caseinomacropeptide by Graphitized Carbon Liquid Chromatography–Electrospray Ionization Ion Trap Tandem Mass Spectrometry Enriqueta Casal & Rosa Lebrón-Aguilar & Yuan Chuan-Lee & Tomiya Noboru & Jesús Eduardo Quintanilla-López
Received: 9 May 2012 / Accepted: 31 July 2012 / Published online: 16 August 2012 # Springer Science+Business Media, LLC 2012
Abstract Isolation and characterization of oligosaccharides from caseinomacropeptide (CMP) are important in understanding the biological and functional properties of CMP. However, it is difficult to achieve this goal, due to the high degree of isomerism present in these types of compounds. In this study, the sialylated oligosaccharides derived from ovine and caprine CMP were released as oligosaccharide alditols by reductive β-elimination and subsequently separated and characterized using graphite carbon column liquid chromatography–negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(−)-MSn). Although, the chromatographic resolution of isomeric oligosaccharides was not achieved perfectly, the characteristic tandem mass spectra of these compounds
allowed differentiating and confirming unequivocally the structure of each one of the oligosaccharides. In CMP of both species, four trisaccharides and four tetrasaccharides were identified as O-glycans. Their chemical structures were identified to be Galβ1-3(NeuAcα2-6)GalNAcol, NeuAcα23Galβ1-3GalNAcol, Galβ1-3(NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3GalNAcol, NeuAcα2-3Galβ1-3 (NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3(NeuAcα26)GalNAcol, NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAcol, and NeuGcα2-3Galβ1-3(NeuGcα2-6)GalNAcol. The LC/MSn methodology using an ion trap-type mass analyzer shown in this study is of general applicability for determination of short O-glycan oligosaccharides. Keywords Caseinomacropeptide . Sialylated oligosaccharides . Isomers . Graphite carbon column . Tandem mass spectrometry
E. Casal Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK R. Lebrón-Aguilar Instituto de Química-Física “Rocasolano”, CSIC, Serrano 119, 28006 Madrid, Spain Y. Chuan-Lee : T. Noboru Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA J. E. Quintanilla-López (*) Instituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain e-mail: [email protected]
Introduction Caseinomacropeptide (CMP), one of the major components of sweet whey, is produced by enzymatic cleavage of κ-casein during cheese production (Manso and López-Fandiño 2004). CMP naturally exists in several forms due to extensive post-translational modifications, such as glycosylation by O-glycosidic linkage, phosphorylation of certain serine residues, and genetic variance (Rasmussen et al. 1997; Moreno et al. 2000, 2001a; Jann et al. 2004; Ceriotti et al. 2004). Various biological and functions of bovine and ovine CMP, including prevention of viral and bacterial infection
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