title: Characterization of heat-induced lactosylation products in caseins by immunoenzymatic and mass spectrometric methodologies.
abstract: Nonenzymatic glycosylation of proteins occur during milk thermal treatment through the Maillard reaction. Immunoenzymatic and spectrometric methodologies were used to investigate caseins modification in samples submitted to different processing. As expected, protein-bound carbonyl content in raw and thermal-treated milks was positively correlated with the severity of the treatment. When immunoblotting and ELISA experiments were carried out either on isolated or whole milk proteins, carbonyl accumulation on caseins and macromolecular aggregates produced by thermal processing was evidenced. A comparative electrospray-mass spectrometry (ES-MS) analysis of different milks allowed verifying the extent of Amadori adducts on the more abundant protein components (alphas1- and beta-casein (alphas1- and beta-CN)). A combined matrix-assisted laser desorption ionization (MALDI)-MS/Edman degradation approach on enzymatic digests identified caseins modification sites. In moderately heat-treated milks, we observed that lactosylation occurred specifically at Lys-34 (alphas1-CN) and Lys-107 (beta-CN); whereas, samples subjected to more severe conditions presented modification at Lys-7, Lys-34, Lys-83, Lys-103, Lys-105, Lys-132 and Lys-193 (alphas1-CN) and at Lys-32, Lys-48, Lys-107, Lys-113 and Lys-176 (beta-CN). Differences in secondary structure of these components was assayed by circular dichroism (CD) spectroscopy.
authors.affiliation: Proteomics and Mass Spectrometry Laboratory, I.A.B.B.A.M., National Research Council, Naples, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Valentina
authors.initials: V
authors.lastname: Perillo
authors.forename: Paolo
authors.initials: P
authors.lastname: Franco
authors.forename: Elena
authors.initials: E
authors.lastname: Fedele
authors.forename: Raffaele
authors.initials: R
authors.lastname: Froio
authors.forename: Lino
authors.initials: L
authors.lastname: Ferrara
authors.forename: Paolo
authors.initials: P
authors.lastname: Bergamo
mesh_terms: D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006031:Glycosylation; D006358:Hot Temperature; D007124:Immunoenzyme Techniques; D007785:Lactose / Q000378:metabolism*; D013058:Mass Spectrometry; D008892:Milk / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
chemical_list: D002364:Caseins; D007785:Lactose