Immunogenic protein profiling of Giardia duodenalis via OFFGEL™ separation and LC-MS/MS

Giardia duodenalis exists in two life cycle stages: the proliferative trophozoite and the resistant, infective cyst, which cause giardiasis-a diarrheal disease with potential long-term effects such as malnutrition, anemia, and stunting, particularly in children. Despite the availability of various diagnostic methods, their limitations result in a 25 % rate of inaccurate treatments. To address this challenge, gaining insights into the protein composition and immunogenicity of G. duodenalis is essential for developing precise diagnostic tools and effective therapies. This study introduces a comprehensive workflow for characterizing Giardia proteins by integrating improved separation technique with an immunoproteomic approach. This includes in vitro cultivation, protein extraction, and OFFGEL™ electrophoresis for enhanced protein separation. Detailed immunoproteomics analysis of trophozoite and cyst proteins, utilizing techniques such as Western blot and mass spectrometry, identified two immunogenic proteins-SH2_2 domain protein and non-specific serine/threonine protein kinase-that are reactive to IgA and IgG antibodies. B-cell epitope prediction (BepiPred 2.0, ABCPred) and antigenicity assessment (VaxiJen 2.0) identified eight target epitopes for the SH2_2 domain protein and four for the kinase, with MHC binding efficiency analyzed using MHCPred. These findings provide insights into Giardia protein immunogenicity, advancing prospects for diagnostic and therapeutic development or giardiasis.