Reproducible and efficient separation of aggregatable zein proteins by CZE using a volatile background electrolyte

Abstract

Zein proteins are a complex mixture of polypetides that belong to the alcohol-soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes their analysis by any analytical method very difficult. Thus, CZE of these proteins requires the use of very complex BGEs noncompatible with online ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, ACN, formic acid and ammonium hydroxide. Besides, it is shown that zein analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a BGE with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-fold in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400 000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.

Time of flight versus ion trap MS coupled to CE to analyse intact proteins

“…Electrospray operating conditions were optimised by adjusting the needle counterelectrode distance in each instrument. The optimum nebuliser/drying gas conditions as optimised in previous works [16,17] were a pressure of 2.76610 4 Pa and 4 L/min of nitrogen at 1208C. A sheath liquid composed of water/2-propanol (50:50 v/v) was used, and delivered at a flow rate of 0.290 mL/h.…”

“…Our group recently provided new analytical methods to study zein proteins by CE-UV [16] and CE-ESI-MS [17]. Zeins are relatively easy to extract from ground maize [18,19], and are commercially available as high quality standard.…”

“…The separation conditions and ionization parameters will be the same as the optimum ones obtained in previous works [16,17]. Thus, the effect of different MS instrumental settings, protein concentrations and type of samples will be investigated on the MS spectra of zein proteins previously separated by CE.…”

“…Both instruments will be compared in terms of LOD, number of proteins detected, and precision and repeatability in the determination of their molecular mass. The CE separations and ESI parameters used in this work will be the same as the ones optimised and described elsewhere [16,17]. Finally, the selected analytical conditions will be applied to study the zein protein fraction from different real samples, namely, wild and transgenic maize, with the aim of identifying possible differences between both varieties.…”

Modification of Resolution in Capillary Electrophoresis for Protein Profiling in Identification of Genetic Modification in Foods

“…[14][15][16] Recently, there were several attempts to employ CE to obtain the protein profiles in extracts of maize and soybean, oriented towards finding some differences between genetically modified plant material or foods and natural variants and their food products. For this purpose both CE measurements with UV 17,18 and MS detections 19,20 were reported.…”