title: A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-Chip coupled with Ion Trap mass spectrometry.
abstract: 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.
authors.affiliation: Department of Endocrinology, Laboratory of Food Chemistry and Mass Spectrometry, University of Milan, Milan, Italy. francesca.brambilla@unimi.it
authors.forename: Francesca
authors.initials: F
authors.lastname: Brambilla
authors.forename: Donatella
authors.initials: D
authors.lastname: Resta
authors.forename: Ilena
authors.initials: I
authors.lastname: Isak
authors.forename: Marco
authors.initials: M
authors.lastname: Zanotti
authors.forename: Anna
authors.initials: A
authors.lastname: Arnoldi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000704:Analysis of Variance; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019295:Computational Biology; D030562:Databases, Protein; D031297:Lupinus / Q000378:metabolism*; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D010455:Peptides / Q000032:analysis; D014674:Plant Proteins, Dietary / Q000032:analysis; D040081:Protein Array Analysis / Q000379:methods; D012015:Reference Standards; D055314:Seed Storage Proteins / Q000032:analysis*; D012639:Seeds / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides; D014674:Plant Proteins, Dietary; D055314:Seed Storage Proteins; C015575:vicilin protein, plant