Evolution of seed allergen quantification – From antibodies to mass spectrometry

Development of accurate, high-throughput approaches for protein allergen quantification is important for the seed industry as a means to monitor natural variability in expression and ensure introduced transgenes do not collaterally alter the expression of any known allergen. Analytical approaches for protein quantification have undergone a renaissance in recent years with the emergence of soft-ionization approaches and advanced mass spectrometers capable of achieving low attomolar sensitivity. These advances coupled with bioinformatic tools to mine mass spectral data are collectively referred to as proteomics, and allow for the large-scale study of proteins with high precision and quantitative accuracy. In this review, we discuss differential and quantitative proteomics workflows that proceed from discovery profiling to targeted, quantitative analysis of specific proteins using stable isotopically-labeled, synthetic peptide doping standards. These synthetic peptide standards, also referred to as AQUA peptides, are synthetic mimics to proteotypic peptides and allow for absolute quantification of proteins in complex biological mixtures. The approaches discussed herein are ideal for the analysis of prominently expressed proteins such as protein allergens from plant seed, as no gels or sample pre-fractionation is required. We discuss these new techniques in the context of traditional, antibody-based technologies for allergen detection and quantification.