Solid-state glycation of beta-lactoglobulin monitored by electrospray ionisation mass spectrometry and gel electrophoresis techniques.
Glycation of beta-lactoglobulin (beta-Lg) with either lactose or galactose in a solid-state medium was monitored using gel electrophoresis techniques and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The kinetics of glycation monitored by SDS polyacrylamide gel electrophoresis showed a molecular weight increase over time of the beta-Lg bands for both sugars, but no significant amounts of aggregated proteins were observed. The isoelectric point of the protein, observed by isoelectric focusing gel electrophoresis, was dramatically affected by galactosylation. LC/MS measurements of beta-Lg variants A and B, over the whole glycation reaction time, showed a larger extent of glycation with galactose (from 4 up to 22 adducts) as compared with lactose (from 0 up to 14 adducts), and confirmed that early Maillard reaction products were the main species observed. Based on the relative abundances obtained from the deconvoluted mass spectra after a 8 h 15 min incubation time at 60 degrees C, the mean values of lactose and galactose molecules bound to the protein species were calculated to be 10.4 and 17.9, and 10.5 and 18.6, for variants A and B, respectively. Furthermore, the charge state distribution data obtained by ESI-MS was studied using different methanol percentages, and indicated that adduct formation with lactose, but more significantly galactose, tends to improve the stability properties of the native protein towards denaturation.
Fenaille
François
F
Nestlé Research Center, Nestec Ltd., Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland.
Morgan
François
F
Parisod
Véronique
V
Tabet
Jean-Claude
JC
Guy
Philippe A
PA
D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000737:chemistry; D006031:Glycosylation; D006358:Hot Temperature; D007525:Isoelectric Focusing; D007782:Lactoglobulins / Q000032:analysis* / Q000378:metabolism*; D007785:Lactose / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013995:Time; D014867:Water / Q000737:chemistry
D007782:Lactoglobulins; D014867:Water; D007785:Lactose; D005690:Galactose