LUPIN PROTEINS AS FOOD INGREDIENTS: DEVELOPMENT AND OPTIMIZATION OF LABEL-FREE METHODS FOR THE QUANTITATIVE ANALYSIS IN MASS SPECTROMETRY

Proteomic techniques offer a new approach for the characterization of food ingredients. Food quality is, in fact, dependent on the presence of bioactive proteins which could have either beneficial or negative effects on human health. For example, quantitative proteomics based on mass spectrometry has been used for detecting and quantifying allergenic proteins or bioactive compounds in tiny amounts. In order to profile the differential protein expression in different samples, two main approaches are reported in literature: stable isotope labeling (SIL) techniques and stable isotope label-free (SIF) techniques. In food analysis, in particular, SIL techniques appear to be too expensive, whereas proteomic tools based on SIF techniques may find some important applications owing to their simple experimental workflows and capability of comparing an unlimited number of samples. In the study 1, an internal standard label-free method based on ion intensity for the simultaneous identification and relative quantification of target storage proteins in total protein extracts (TPEs) of the seeds of Lupinus albus (white lupin) was developed. The use of an innovative microfluidic system, the HPLC-Chip, coupled with a classical Ion Trap mass spectrometer has enabled a complete qualitative characterization of all seed storage proteins in a single analysis of the TPE tryptic digest. The differential analyses of gamma-conglutin, a mature protein, and of the vicilins, a complex protein class, in four lupin cultivars were performed optimizing two suitable bioinformatics parameters, the “normalized protein average of common reproducible peptides” (N-ACRP) and the “normalized protein mean peptide spectral intensity” (N-MEAN), respectively. It is important to underline that a relative approach does not enable the absolute quantification of the target protein in the sample and, consequently, lacks to give a real evaluation of the potential bioactivity of the food. The quantitative approaches AQUA and QConCAT, requiring the chemical synthesis of all isotope-labeled peptides, appear not suitable to their application in the field of food chemistry. Moreover, in food analysis the isotope labelling techniques appear to be too expensive. In order to achieve a real absolute quantification of the lupin gamma-conglutin, in study 2, a very selective method was develop and applied to TPE. The Multiple Reaction Monitoring (MRM) label-free absolute quantitative method, based on the “standard addition” strategy was developed with the target to absolutely quantify the lupin target protein, i.e. gamma-conglutin, in the flour of white lupin. The four main features of the method are the following: a) the chromatographic separation was performed on a very efficient HPLC-Chip system coupled with a ion trap mass spectrometer; b) five proteotypic peptides of gamma-conglutin were selected and analyzed with a Multiple Reaction Monitoring (MRM) method; c) the absolute quantification was obtained by the standard addition approach by purifying gamma-conglutin from lupin seed; d) the matrix effect was overcome by the addition of an exogenous protein.