Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Abstract

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.

Physicochemical characterisation of kafirins extracted from sorghum grain and dried distillers grain with solubles related to their biomaterial functionality

“…1 . For zein, the banding pattern was in line with those previously reported 22 , 24 , 25 . The zein shows a band at ~ 19 kDa corresponding to β polypeptide monomer, a band at ∼ 21–23 kDa corresponding α 1 and α 2 polypeptides.…”

Physicochemical characterisation of kafirins extracted from sorghum grain and dried distillers grain with solubles related to their biomaterial functionality

“…1. For zein, the banding pattern was in line with those previously reported18,20,21 . The zein shows a band at ~ 19000 KDa corresponding to β polypeptide monomer, a band at ∼ 21,000-23,000 KDa corresponding α 1 and α 2…”

Nitrogen Storage in Crops: Case Study of Zeins in Maize

“…When analyzing zein proteins, different extraction procedures are applied: wide range of aqueous ethanol solutions [20][21][22] or aqueous isopropanol solutions [23,24], highly concentrated alkali solutions (pH 11 or above) [25], highly concentrated aqueous urea solutions (8 M) [26], or anionic detergent-containing solutions [20,26,27]. The extractions are performed on wide range of extraction temperatures, ranging from 25 to 130°C [20,21,26], with or without addition of reducing agents as 2% 2-mercaptoethanol [27] or 10 mM DTT in 25 mM ammonium hydroxide [22]. Solubility-enhancing ingredients, such as 0.0125 M sodium borate [27], 0.5% sodium hydroxide [28], 0.5% sodium bisulfite [24] are also often added.…”

“…Hence, analysis of zein extracts is complex and include the application of various techniques often used in protein separations. Combination of electrophoresis methods, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), which only provides the molecular weights (MW) [22,27,29,30], or the two-dimensional gel electrophoresis (2DE), which provides insight into MW and charge [22], with or without the mass spectrometry (MS) for protein identification [22,31], are used in zein analysis. Capillary electrophoresis (CE), the electrophoretic method also used for zein separation, has enabled the identification of more fractions by MS [32].…”

“…Liquid chromatography-mass spectrometry (LC-MS), a method widely used in proteome analysis due to its high sensitivity, was for the first time adopted for zein evaluation in 2020; the authors coupled multi-enzyme digestion with nano-LC-MS/ MS [35]. Separation techniques mentioned here are used for the analysis of protein compositions in biological samples of high complexity [22]. Regardless of numerous approaches in zein analysis, the most suitable method will be selected based on desired trait of maize zein proteins.…”