Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

This study improves LC-MS-based trace level peanut allergen quantification in processed food by refining method robustness, total analysis time and method sensitivity.  Extraction buffer (six compared) and peptide choice were optimised and found to profoundly affect method robustness (Fig. 1A). A rapid extraction and in-solution digestion method omitting subsequent reduction, alkylation and sample clean-up steps was found to outperform a classic FASP protocol for a set of selected proteotypic peanut allergen peptides (Fig. 1B). This optimisation resulted in reducing the total analysis time from the previously reported ~5.5-20 hours to ~2.5 hours only. The three best functioning peptides were used to quantify peanut allergen in five peanut varieties from different geographical origin that underwent various types of food processing (including oil and air roasting) and excellent robustness was obtained (Fig. 1C). Accurate quantification (CVs <15%) with matrix-matched calibration curves (R²=0.99-0.97; Fig. 1D) was equally achieved for peanut muffin and ice-cream with excellent linearity (0.25-1000 ppm). Moreover, the best performing peptide enabled excellent recovery rates in ice-cream (106.0 ± 15.1%) and peanut muffin (72.7 ± 13.4%). Sensitivity (LOD=0.25-0.5 ppm; LOQ=0.5-1.0 ppm) was 2- to 20-fold improved compared to previous methods depending on the peptide. These methodological improvements contribute to robust peanut detection in food and can be translated to additional food-borne allergens.

Fig. 1 A) Optimisation of buffer (six buffers compared) and peptide choice. B) Peak areas obtained for one proteotypic peanut allergen peptide using microwave assisted in-solution tryptic digestion at varying incubation times and temperatures. Dashed line indicates peak area obtained using a ~20 h FASP protocol. C) Peak areas obtained using the rapid method for different peanut varieties that underwent various food processing treatments. D) A matrix-matched (muffin) and buffer calibration curve for one of the three best performing peptides for peanut allergen quantification.