[show abstract][hide abstract] ABSTRACT: Sialylated O-linked oligosaccharides are involved in many biological processes, such as cell-cell interactions, cell-substance adhesion, and virus-host interactions. These activities depend on their structure, which is frequently determined by tandem mass spectrometry. However, these spectra are frequently analyzer-dependent, which makes it difficult to develop widely applicable analytical methods. In order to deepen the origin of this behavior, two couples of isomers of sialylated O-linked oligosaccharides, NeuAc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuAc alpha2-6)GalNAc-ol and NeuGc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuGc alpha2-6)GalNAc-ol, were analyzed by liquid chromatography/negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(-)-MS(n)) using both an ion trap and a triple quadrupole mass spectrometer. Results clearly showed that while ions obtained in the triple quadrupole instrument fitted very well with the standard fragmentation routes, in the ion trap several intense ions could not be explained by these rules, specially a fragment at m/z 597. Furthermore, this ion was observed in the mass spectrum of those isomers that sialic acid binds to GalNAc by an alpha2-6 linkage. From the MS(3) spectrum of this ion an unexpected structure was deduced, and it led to propose alternative fragmentation pathways. Molecular mechanics calculations suggested that the found atypical route could be promoted by a hydrogen bond located only in alpha2-6-linked oligosaccharides. It has also been demonstrated that this process follows a slow kinetic, explaining why it cannot be observed using an ion beam-type mass analyzer. In conclusion, ion traps seem to be more appropriate than triple quadrupoles to develop a reliable analytical method to distinguish between isomeric O-linked glycans.
Rapid Communications in Mass Spectrometry 03/2010; 24(7):885-93. · 2.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid and sensitive analysis of glycans is essential for glycomics. We previously reported an apparatus, the AutoGlycoCutter (AGC), for rapid release of O-linked glycans under alkaline conditions and its application to rapid analysis of glycans in proteoglycans. We now report an application of the AGC to obtain mucin-type glycans with reducing end (i.e., hemiacetal group) within only 3 min. The released oligosaccharides could be labeled with fluorescent 2-aminobenzoic acid for analysis by normal-phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We could detect O-glycans from as low as 5 pmol of bovine caseino glycomacropeptide (CGMP) by the proposed procedures. The validity of the current method was shown by the analyses of the released O-glycans from some standard glycoproteins: bovine submaxillary mucin, bovine fetuin, porcine stomach mucin, and human colostrum immunoglobulin A. The advantage of the current method was also demonstrated in comparative analysis of mucin-type glycans in CGMP derived from three different animal species.
[show abstract][hide abstract] ABSTRACT: A simple, economical and non-toxic method is described for the solubilization of undenatured β-lactoglobulin (β-lg) from complexes with chitosan. The effect of pH (8–10), ionic strength (0.08–0.3 m) and volume ratio between sodium acetate solutions and whey on the dissociation of β-lg–chitosan complexes was evaluated. Following a single extraction step with the addition of 10 mL of 0.1 m sodium acetate solution at pH 9 to the β-lg–chitosan complexes obtained from 1 mL of cheese-whey, a recovery of 90% of β-lg with a protein purity of 95% was achieved, suggesting that electrostatic interactions play a key role in the complexation of β-lg with chitosan. The presence of free chitosan in solution was ruled out according to gas chromatography with flame-ionization detector analysis after acid hydrolysis. NMR spectroscopy showed that the recovered β-lg after further dialysis had structural features very similar to the native protein.
[show abstract][hide abstract] ABSTRACT: A method is described for selective removal of undenatured beta-lactoglobulin from cheese whey based on interactions between whey proteins and chitosan. Whey was previously clarified at pH 4.5 with addition of chitosan (25 mg/100 mL), and selective removal of beta-lactoglobulin was studied in the pH interval 4.6 to 6.5. Addition of chitosan caused selective precipitation of beta-lactoglobulin that increased with pH. The content of beta-lactoglobulin in whey decreased as the amount of chitosan added was increased. At pH 6.2, addition of 1.9 to 3.0 mg/mL of chitosan led to complete removal of beta-lactoglobulin, whereas at least 80% of the rest of whey proteins remained in solution. The production of cheese whey without beta-lactoglobulin could help to expand the applications of dairy by-products in food processing, and to isolate hypoallergenic whey protein concentrates.
Journal of Dairy Science 06/2006; 89(5):1384-9. · 2.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: A method is described for selective removal of unde- natured β-lactoglobulin from cheese whey based on in- teractions between whey proteins and chitosan. Whey was previouslyclarified atpH 4.5with additionof chito- san (25 mg/100 mL), and selective removal of β-lacto- globulin was studied in the pH interval 4.6 to 6.5. Addi- tionof chitosancaused selectiveprecipitation of β-lacto- globulin that increased with pH. The content of β- lactoglobulin in whey decreased as the amount of chito- san added was increased. At pH 6.2, addition of 1.9 to 3.0 mg/mL of chitosan led to complete removal of β- lactoglobulin, whereas at least 80% of the rest of whey proteins remained in solution. The production of cheese whey without β-lactoglobulin could help to expand the applications of dairy by-products in food processing, and to isolate hypoallergenic whey protein concen- trates.
Journal of Dairy Science - J DAIRY SCI. 01/2006; 89(5):1384-1389.
[show abstract][hide abstract] ABSTRACT: The effect of supercritical carbon dioxide treatment on the development of the Maillard reaction in powdered model systems of lactose/ovine caseinmacropeptide and lactose/β-lactoglobulin at different pH values was studied. Supercritical carbon dioxide treatments in static conditions at 30 MPa and 50 °C for up to 5 h were applied to model systems. Control experiments at 50 °C were also performed. All assayed model systems treated with carbon dioxide under supercritical conditions showed lower extent of the Maillard reaction than control treated samples. Differences between supercritical carbon dioxide treated and control samples increased with pH. These results indicate that supercritical carbon dioxide treatment of food samples does not favour the Maillard reaction and thus can be applied in foods that may require special care to avoid excessive loss of available lysine.
[show abstract][hide abstract] ABSTRACT: The use of chitosan, a partially deacetylated chitin, to fractionate aqueous solutions of caseinmacropeptides (CMPs) was studied. The polycationic character of chitosan at acidic pH values allows the formation of complexes with negatively charged CMP molecules, inducing their flocculation. Glycosylated CMP (GMP) has higher affinity for chitosan than nonglycosylated forms (NGMP). The carboxylic groups in the carbohydrate moiety of the GMP increase the negative charge of the molecule and may play a role in the selective precipitation. At pH 5.0, 0.08 mg/mL of chitosan completely removed the GMP whereas 70% of NGMP remained in solution. As the pH increased, the amount of chitosan to ensure complete removal of GMP increased up to 0.19 and 0.34 mg/mL for pH 6.0 and 6.6, respectively.
Journal of Agricultural and Food Chemistry 03/2005; 53(4):1201-4. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycation of caseinmacropeptide (CMP) during storage with lactose at 40 and 50 °C and water activity 0.33–0.65 was studied by measurement of furosine. At pH 8.0 and aw0.44, maximum levels of furosine up to 1.9 mg/100 mg CMP were obtained within five days at 50 °C, while this value had not been reached at 40 °C after 13 days of storage. Increasing pH up to 11 caused considerable increase in the rate of furosine formation and maximum values were obtained after 9 h at 50 °C. The rate of furosine formation was also enhanced with increasing aw up to 0.65. These results showed that lactosylated CMP can be efficiently prepared during short time storage of CMP–lactose mixtures under appropriate pH, water activity and temperature conditions.
[show abstract][hide abstract] 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, NeuAcα2-3Galβ1-3GalNAcol, Galβ1-3(NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3GalNAcol, NeuAcα2-3Galβ1-3(NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3(NeuAcα2-6)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.