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.
Steps to reproduce
There are three roots for reproducing out results. First, you may check "Data Dependent Acquisition" file and utilize this raw file for identifying walnut candidate marker peptides using Proteome DiscovererTM version 2.2. Next, through data file "Parallel Reaction Monitoring", you can verify specificity confirmed candidate marker peptides using Skyline software. Finally, you can get validation results from data file "Validation using Multiple Reaction Monitoring".