Enhanced identification of glycoproteins and allergens in Hevea brasiliensis latex using innovative enrichment techniques.

Hevea brasiliensis is the most efficient crop for latex production and has been the main source for global commercialisation since 1876. However, little is known about its glycosylation properties. Studying glycoproteomics has been challenging due to analytical difficulties and the heterogeneous nature of glycans. In this study, we extracted natural rubber latex (NRL) proteins, purify peptide/glycopeptide mixtures and isolate glycopeptides using HILIC, SAX, and TiO₂ enrichment methods. TiO₂ showed high selectivity compared to SAX and HILIC, retaining unique proteins and glycans based on hydrophilicity. We identified proteins using LC-MS/MS and PEAKS Studio 7.5. These methods significantly improved protein coverage compared to non-enriched samples. Three glycoproteins (patatin, REF, and glucan endo-1,3-beta-D-glucosidase) and seven internationally recognised allergens (hev b 1, hev b 2, hev b 3, hev b 5, hev b 7, hev b 14, and hev b 15) were identified. These enrichment methods not only helped identify glycoproteins but also improved protein sequence coverage.