Separation of Intact Cashew Nut Proteins by Reversed-Phase Liquid Chromatography for Improved Allergen Resolution

Intact cashew nut allergenic proteins were separated using a reversed-phase liquid chromatography (RP-HPLC) method to improve resolution over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Cashew proteins were successfully extracted with solutions containing SDS or guanidine hydrochloride (Gdn-HCl) under reduced and nonreduced conditions. Through evaluation by SDS-PAGE and RP-HPLC, reduced extracts were shown to be more stable over 14 days than nonreduced extracts. Major allergens (Ana o 2 and Ana o 3) were identified in the RP-HPLC method by fraction collection and confirmed by SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The RP-HPLC method was repeatable (peak area %RSD range: 0.5-1.0%) and showed good linearity of peak groups (R² range: 0.9991-0.9998) for potential usage for quantitation. The methods were applicable to cashew flour, milk, and butter samples as well as recombinant Ana o 3, demonstrating profiling or fingerprinting abilities for cashew products with varied genetics and forms.