Peptide-Based Quantitative Analysis of Rapeseed Storage Proteins and Their Isoforms by Targeted LC-MS/MS

Rapeseed (Brassica napus) is an underutilized source of plant protein in the food industry, with napin and cruciferin constituting its two major seed storage proteins. Quantitation of these proteins and their isoforms is essential for understanding protein composition in rapeseed-derived protein ingredients and optimizing their functional applications. We developed a selected reaction monitoring LC-MS/MS method with surrogate matrix-matched calibration for the peptide-based quantitative analysis of both polypeptide chains in each of seven napin and six cruciferin isoforms. The method had an LLOQ of 0.60-29 nM, but the accuracy for cruciferin for the two chains differed due to trypsin hydrolysis efficiency variation, The method was applied to napin and cruciferin isolates, rapeseed protein extract, and commercial rapeseed protein isolate. Quantitative analysis revealed distinct protein compositions and isoform distributions across samples, demonstrating the capability to resolve protein heterogeneity. This targeted LC-MS/MS approach provides a useful tool for the characterization of rapeseed protein samples.