Mass spectrometry-based proteomics pipeline for screening hazardous proteins in alternative protein-based foods

Alternative protein-based foods (APBFs) are increasingly preferred globally. However, the assessment of their safety, especially protein safety, is limited. Advanced mass spectrometry (MS)-based proteomics is a powerful tool to provide comprehensive protein information and identify abnormal proteins/peptides. Here, we present an effective non-targeted workflow for screening hazardous proteins in APBFs that includes a protocol for efficient sample preparation, an optimized MS approach, and a data analytics pipeline with a comprehensive hazardous protein database. Of note, protein extraction was largely optimized, which facilitated not only the detection of harmful proteins, but also the protein profiling of complex food matrix. Nine APBFs were tested, and 45 proteins in 6 APBFs were identified to be potentially hazardous. A targeted MS assay was developed, which confirmed the presence of 11 selected hazardous proteins at femtomole sensitivity. This pipeline can be positioned as a rapid approach for tracking emerging protein hazards in various protein-based foods.