Premium
Solid‐state glycation of β‐lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques
Author(s) -
Fenaille François,
Morgan François,
Parisod Véronique,
Tabet JeanClaude,
Guy Philippe A.
Publication year - 2004
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.539
Subject(s) - chemistry , glycation , chromatography , lactose , mass spectrometry , maillard reaction , galactose , electrospray ionization , lysine , matrix assisted laser desorption/ionization , amino acid , biochemistry , organic chemistry , desorption , receptor , adsorption
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 β‐lactoglobulin (β‐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, Leu 1 (NH 2 terminal) and the Arg 124 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 β‐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 (Arg 40 , Arg 124 and Arg 148 ) that were modified, and also dialkylations on specific lysine residues (Lys 47 , Lys 75 ). Copyright © 2003 John Wiley & Sons, Ltd.