Chromatography–Mass Spectrometry Analysis of Plant Protein Concentrates

Abstract

This work was devoted to a study of the composition of protein concentrates from amaranth grain (Amaranthus hypochondriacus L.) of variety Voronezh. Amaranth protein concentrates were obtained by alkaline extraction of proteins and neutralization of the solution followed by ultrafiltration, by separating the starch fraction with amylolytic enzymes, and by alkaline extraction of proteins and their precipitation at pH 4.5. Conditions for the extraction of proteins followed by chromatography–mass spectrometric analysis and identification were selected. It was found that proteins from amaranth grain were more effectively extracted with a buffer with urea at protein concentrations of 1.7, 1.9, and 2.9 mg/cm³ in solution, respectively, while a buffer with detergents was more effective for the extraction of low-molecular-weight proteins at protein concentrations of 4.9, 2.9, and 9.0 mg/cm³ in solution, respectively. As a result of HPLC–MS/MS analysis followed by identification and search in the UNIPROT database, it was established that the main protein of amaranth grain is 11S-globulin, which acts as a reserve protein of amaranth seeds. In amaranth concentrates, 14 unique proteins belonging only to A. hipochondriacus L. were identified, and also proteins that did not belong to this species were reliably identified. Based on the results of semiquantitative analysis of the peptide profile of amaranth grain protein concentrates, a high frequency of occurrence of the main 11S-globulin proteins was established in all samples. The frequency of occurrence of other proteins in samples obtained by different methods differed significantly due to the peculiarities of protein isolation from amaranth grain. The results obtained can be used to prepare plant protein concentrates with a given protein composition.