A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-Chip coupled with Ion Trap mass spectrometry.

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label-free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label-free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC-Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (beta-conglutin precursor and vicilin-like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin-encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the "normalized protein average of common reproducible peptides" (N-ACRP) for gamma-conglutin, which is a homogeneous protein, and the "normalized protein mean peptide spectral intensity" (N-MEAN) for the highly heterogenous class of the vicilins.

Preliminary approaches for the development of a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the detection and label-free semi-quantitation of the main storage proteins of Lupinus albus in foods.

Since recent literature has indicated that white lupin (Lupinus albus) may be a useful source of hypocholesterolemic proteins to be used in functional food formulation, our final goal is the development of a fast and automated high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the detection and the label-free semi-quantitation of the main lupin globulins in lupin foods and food ingredients. We present here some preliminary results in this direction. As a first step a total protein extract (TPE-WF) from lupin flakes was pre-fractionated by anion-exchange chromatography and each fraction was digested with trypsin and analyzed by HPLC/ESI-MS/MS. Subsequently, the tryptic digest of TPE-WF was directly analyzed by HPLC/ESI-MS/MS without any pre-fractionation. Eventually, in order to test the applicability of the method to real samples, a lupin beverage and two lupin protein isolates were analyzed. Both Mascot and Spectrum Mill MS Proteomics Workbench software were used to identify the protein composition in these samples and Spectrum Mill was used also to test the possibility of developing a label-free semi-quantitation method based on peptide hits. Encouraging results were obtained especially in the detection of the hypocholesterolemic component beta-conglutin.