First Ultraperformance Liquid Chromatography Based Strategy for Profiling Intact Proteins in Complex Matrices: Application to the Evaluation of the Performance of Olive (Olea europaea L.) Stone Proteins for Cultivar Fingerprinting

Abstract

There is a clear need for accelerating protein separations by HPLC. Different proposals have been developed including the use of perfusion and monolithic stationary phases. Nevertheless, these stationary phases, in some occasions, do not provide enough efficiency to resolve these large molecules when they are present in complex matrices. Although ultraperformance liquid chromatography (UPLC) columns have been successfully used for the efficient and rapid separation of small molecules, this is the first time these columns were proposed for the separation of intact proteins in a real complex matrix: the olive stone. Two different strategies were employed for the extraction of olive proteins: enzymatic assisted extraction and buffered extraction. Five different columns traditionally employed for the separation of proteins were used, and results were compared with those obtained when using different sub-2 microm particle columns. Separations obtained with sub-2 mum particle columns significantly improved the separations obtained with the other columns. This paper also demonstrates the applicability of protein profiles obtained from the olive stone for the discrimination among olive varieties.