Investigation of the Lactosylation of Whey Proteins by Liquid Chromatography−Mass Spectrometry

Abstract

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

“…β-Lactoglobulin (β-LGB) and α-lactalbumin (α-LAB) have been identified as main protein reaction targets in dairy products [22]. Their quantitative lactosylation, generally assessed by various MS procedures, has been widely used to monitor milk quality and thermal history [22][23][24][25][26][27][28][29][30][31]; for example, it has been demonstrated that the lactosylated protein forms account for almost 3, 30 and 70% of the β-LGB content in pasteurized, UHT and dry infant formula samples, respectively. Caseins (CSNs) have also been reported as lactosylated species depending on milk thermal treatment [32].…”

Non-Enzymatic Degradation Pathways of Lactose and Their Significance in Dairy Products

“…Additional analysis by mass spectrometry suggested the increases in mass for the whey protein peaks were approximately multiples of 320 Da consistent with incremental lactosylation reactions. Other studies have demonstrated that lactosylation of -lactoglobulin can proceed under relatively mild conditions in the absence of browning is site specific (appearing to prefer Lys 47 ), and results in conformational changes and ultimately aggregation via non-covalent interactions (Leonil et al, 1997;Morgan et al, 1999b;Chevalier et al, 2002;French et al, 2002;Czerwenka et al, 2006).…”

Food‐Related Carbohydrate Ligands for Galectins

“…Alternatively, a number or polyclonal and monoclonal antibodies against lactulosamine-modified proteins and polylysine have been developed, which dramatically increased sensitivity of LA analysis by the use of immunodetection techniques (78)(79)(80). More recently, efforts to develop direct instrumental analysis methods for determining lactulosamine modification in dairy products have succeeded in applying the liquid chromatographyÀmass spectrometry to enzymatic hydrolyzates of milk proteins (81,82).…”