Separation and identification of iron-chelating peptides from defatted walnut flake by nanoLC-ESI-MS/MS and de novo sequencing

Walnuts are rich in unsaturated fatty acids and are processed to walnut oil. The by-product (defatted walnut flake) consists of approximately 50% protein and is usually treated as waste or low-value-use material. In this study, walnut protein was isolated and hydrolyzed to low-molecular-weight peptides. Iron-binding walnut peptides were prepared by iron-immobilized affinity chromatography.(IMAC-Fe3+). Some iron-chelated peptides adsorbed to the IMAC-Fe3+ column were easily broken by NaH2PO4, whereas part of the peptides isolated from IMAC-Fe3+ column remained as peptide-iron complexes and could only be disrupted by EDTA-2Na. Subsequently, the iron-binding walnut peptides were separated into approximately 10 fractions by RP-HPLC. The main peak, with retention time of 32 min, was further analyzed by nanoLC-ESI-MS/MS. Two main components in F(32mi)n were characterized as LAGNPDDEFRPQ and VEDELVAVV with de novo sequencing.