Identification and Quantification of ε-(γ-Glutamyl)lysine in Digests of Enzymatically Cross-Linked Leguminous Proteins by High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS)

A rapid and convenient method for the precise quantification of ε-(γ-glutamyl)lysine isopeptide in lyophilized proteolytic digests of cross-linked plant protein samples was developed. The isopeptide was baseline-separated from three other isomers containing lysyl and glutamyl residues by reverse-phase high-performance liquid chromatography after exhaustive proteolytic digestion of the samples cross-linked by a microbial transglutaminase (MTG). Highly selective detection was performed by electrospray mass spectrometry in MS/MS mode. Demonstrating the applicability of the suggested analytical procedure, enzymatic cross-linking of protein isolates from soy [Glycine max (L.) Merr.], pea [Pisum sativum L.], and the sweet lupin species Lupinus albus L. and Lupinus angustifolius L. was investigated after incubation with 0.01 g of MTG/100 g of protein for 0−240 min at 40 °C. The liquid chromatography−mass spectrometry (LC−MS) method was successfully applied to monitor the kinetics of ε-(γ-glutamyl)lysine isopeptide formation. Since the calculated initial levels of ε-(γ-glutamyl)lysine in the genuine leguminous protein isolates were between 40 and 77 μmol/100 g, an isopeptide detection limit of 0.5 μg/mL, corresponding to approximately 50 μmol/100 g of protein, was shown to suffice for quantifying the cross-linking rate enzymatically induced by MTG. Concentrations of ε-(γ-glutamyl)lysine in the texturized proteins ranged from 100 to 500 μmol/100 g of protein.

Keywords: ε-(γ-glutamyl)lysine; transglutaminase; cross-link; leguminous proteins; LC−MS.