HPLC-Chip-Multiple Reaction Monitoring (MRM) method for the label-free absolute quantification of γ-conglutin in lupin: Proteotypic peptides and standard addition method

In food science there is a growing demand of methods for the absolute quantification of proteins, such as allergens or bioactive proteins, and shotgun proteomics based on mass spectrometry is a promising analytical tool in this area. This paper describes an innovative label-free method for the absolute quantification of γ-conglutin, one of the most relevant lupin seed proteins, which is hypoglycaemic and a major allergen. The main features of the method are: (a) the chromatographic separation was performed on an HPLC-chip system coupled with an ion trap mass spectrometer; (b) five proteotypic peptides of γ-conglutin were selected and analysed with a multiple reaction monitoring (MRM) method; (c) absolute quantification was obtained by the standard addition method after purification of a reference sample of γ-conglutin from lupin seed; (d) the matrix effect was overcome by spiking with an exogenous protein, i.e. BSA, as internal standard.