title: Identification and quantification of epsilon-(gamma-glutamyl)lysine in digests of enzymatically cross-linked leguminous proteins by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS).
abstract: A rapid and convenient method for the precise quantification of epsilon-(gamma-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 degrees C. The liquid chromatography-mass spectrometry (LC-MS) method was successfully applied to monitor the kinetics of epsilon-(gamma-glutamyl)lysine isopeptide formation. Since the calculated initial levels of epsilon-(gamma-glutamyl)lysine in the genuine leguminous protein isolates were between 40 and 77 micromol/100 g, an isopeptide detection limit of 0.5 microg/mL, corresponding to approximately 50 micromol/100 g of protein, was shown to suffice for quantifying the cross-linking rate enzymatically induced by MTG. Concentrations of epsilon-(gamma-glutamyl)lysine in the texturized proteins ranged from 100 to 500 micromol/100 g of protein.
authors.affiliation: Fraunhofer Institute for Process Engineering and Packaging, Giggenhauser Strasse 35, 85354 Freising, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schäfer
authors.forename: Michael
authors.initials: M
authors.lastname: Schott
authors.forename: Fritz
authors.initials: F
authors.lastname: Brandl
authors.forename: Sybille
authors.initials: S
authors.lastname: Neidhart
authors.forename: Reinhold
authors.initials: R
authors.lastname: Carle
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004151:Dipeptides / Q000032:analysis*; D007887:Fabaceae / Q000737:chemistry*; D031297:Lupinus / Q000737:chemistry; D018532:Pisum sativum / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D030262:Soybean Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004151:Dipeptides; D010940:Plant Proteins; D030262:Soybean Proteins; C001299:epsilon-(gamma-glutamyl)-lysine; D011503:Transglutaminases