Discovery, Verification, and Validation of Walnut Protein Marker Peptides Using LC-MS Approaches

A key requirement of liquid chromatography-mass spectrometry (LC-MS)-based allergenic food protein analysis methods is to use protein marker peptides with good analytical performances in LC-MS analysis of commercial processed foods. In this study, we developed a multistage walnut protein marker peptide selection strategy involving marker peptide discovery and verification and LC-MS validation of chemically equivalent stable isotope-labeled peptides. This strategy proposed three walnut protein marker peptides, including two new marker peptides. Our LC-MS-based walnut protein analysis method using the three stable isotope-labeled peptides showed acceptable linearity (R2 >0.99), matrix effects (coefficient of variation <±15%), sensitivity (limit of detection >0.3 pg/μL, limit of quantification >0.8 pg/μL), recovery (85.1–103.4%), accuracy, and precision (coefficient of variation <10%). In conclusion, our multistage marker peptide selection strategy effectively selects specific protein marker peptides for sensitive detection and absolute quantification of walnut proteins in LC-MS analysis of commercial processed foods.