Amanda Léa Cavalcante dos Santos¹, Helton Colares da Silva², Renata Pinheiro Chaves¹, Celso Shiniti Nagano¹, Alexandre Holanda Sampaio¹, Rômulo Farias Carneiro¹

Comprehensive structural elucidation of novel proteins from marine organisms remains a major challenge in proteomics, particularly for species lacking genomic resources. In this study, we employed a bottom-up proteomic workflow to characterize lectins from three marine red algae: Solieria filiformis, Meristiella echinocarpa, and Amansia multifida. Protein extracts were resolved by SDS-PAGE, and target bands were excised, followed by in-gel reduction, alkylation, and enzymatic digestion using trypsin and complementary proteases. Peptides were separated by nano-flow reversed-phase chromatography and analyzed on a SYNAPT HDMS (Waters) mass spectrometer operating in data-dependent acquisition mode. Manual de novo interpretation of CID-MS/MS spectra was performed to overcome the limited database coverage typical for algal lectins. To enhance sequence coverage, we employed a dual-enzyme digestion strategy and optimized peptide recovery from gel matrices, addressing key limitations of traditional bottom-up approaches. Intact mass profiling using ESI-MS confirmed the monomeric nature of all three lectins and revealed molecular masses consistent with the theoretical values derived from sequencing (ranging from 27.9 to 28.7 kDa). Full-length sequences were successfully reconstructed for all lectins, each displaying four tandemly repeated CRDs (carbohydrate recognition domains) characteristic of the OAAH lectin family. Conserved binding motifs (QWGGREGPI) were identified, suggesting specificity toward high-mannose glycans. These findings expand the phylogenetic distribution of OAAH lectins and reinforce the value of integrative bottom-up strategies supported by intact mass validation. Our approach addresses common pitfalls such as poor peptide extraction, underrepresentation of hydrophobic regions, and fragmentation inefficiencies, thereby enabling high-confidence annotation of uncharacterized proteins. In addition to structural insights, this work provides the molecular basis for future functional studies of algal lectins in host-pathogen interactions and their potential biotechnological applications.

Agradecimentos: We are immensely grateful to the funding agencies CNPq, CAPES, and FUNCAP for their financial and institutional support, which was fundamental for the development of this research.