Solid-state glycation of beta-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques.
The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75).
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
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid; D005690:Galactose / Q000378:metabolism*; D006031:Glycosylation; D007782:Lactoglobulins / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D007782:Lactoglobulins; D007785:Lactose; D005690:Galactose