Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

An investigation of the physiochemical changes in field pea flour after heat treatment. (#128)

Joshua JH Hutchings 1 , Leigh LD Donnellan 1 , John JC Carragher 2 , Maria MS Saarela 2 , Peter PH Hoffmann 1
  1. Clinical and health sciences, University of South of Australia, Bowden, SA, Australia
  2. South Australian Research and Development Institute (SARDI), Adelaide, SA, Australia

Field peas are a nutritious food source that have shown potential as a functional food ingredient and heat treatment can be used to improve the sensory and functional characteristics of the flour. Changes to the pea proteome before and after heat treatment can be monitored using MS-based techniques, however plant proteins exhibit poor solubility and require an effective extraction protocol in the LC-HRMS (Orbitrap) workflow for satisfactory sequence coverage and protein ID confidence. Evaluation of three unique extraction protocols 50% (v/v) isopropanol (IPA) 50 mM Tris-OH, and a 2-step IPA → 8 M urea protocol (Combo) showed IPA extracted 8.6% protein, Tris-OH 40.6% and Combo 87.1% from raw pea flour (17.2% protein). SDS-PAGE showed IPA enriched low MW proteins (<20 kDa) while Tris-OH and Combo showed clear bands from 20-100 kDa, with Combo depleted of proteins <20 kDa. The number of proteins/peptides identified using LC-HRMS by each extraction protocol were 356/1781 (IPA), 431/2824 (Tris-OH) and 421/2707 (Combo). Heatmap and PCA analysis showed IPA extraction favoured proteins involved in nutrient storage, plant defence and metal ion binding, Tris-OH extracted more cytosolic proteins involved in amino acid synthesis and energy production while Combo tended to extract cell membrane proteins with a higher isoelectric point but did not enrich any specific class. Tris-OH was selected for subsequent proteomic analysis of heat-treated flour samples. To evaluate the effect of heating on the pea flour, samples were heated in a microwave for 5 min or drum roaster for 7 min. SDS-PAGE showed no major changes in protein profile following microwave roasting however, drum roasting showed evidence of protein aggregation and degradation of protein. LC-HRMS analysis showed a significantly lower protein ID count after either heat treatment and a significantly higher missed cleavage rate after drum roasting. Analysis of protein bound Maillard reaction products (MRPs) revealed that microwave roasting caused a ~4-fold increase in modified peptides and drum roasting an ~11-fold increase when compared to the untreated flour. Modifications were predominantly methylglyoxal derived hydroimidazolones (MG-H) within high abundance nutrient storage proteins vicilin and legumin. Proteomic approaches provide a relatively fast and effective tool for determining the need for further quantitative mass spectrometry of MRPs and providing the plant-based food industry with insight into the effect of different heat processing techniques.