Paulo Anderson Paiva Martins¹, Philippe Lima Duarte ¹, Rômulo Farias Carneiro¹, Celso Shiniti Nagano¹, Alexandre Holanda Sampaio¹

Bottom-up is a widely employed strategy in proteomics for large-scale protein identification and characterization. This method is based on the enzymatic digestion of proteins into smaller peptides, which are subsequently separated and analyzed by mass spectrometry. Protein identification is then inferred from the peptide mass spectra, which are compared with known sequence databases or manually analyzed. This approach allows for the identification of hundreds of proteins in a single MS experiment. However, achieving 100% sequence coverage and characterizing intact proteoforms—including post-translational modifications and isoforms—remain inherent challenges of this methodology. In our group, which focuses on the characterization of biologically active proteins from marine invertebrates and macroalgae, we employ a robust methodology based on the bottom-up approach. Initially, protein samples are subjected to SDS-PAGE, after which the bands of interest are excised from the gel. The proteins in these spots are reduced and alkylated, and digestion is performed using trypsin and other proteolytic enzymes to maximize sequence coverage and obtain complementary peptides. The resulting digestion peptides are then introduced into a high-performance liquid chromatography (HPLC) system coupled to a SYNAPT HDMS mass spectrometer (Waters Corp.). A distinctive aspect of our research lies in the peptide sequencing step. Due to the scarcity of marine invertebrate and macroalgal protein sequences in public databases, automatic homology-based sequencing often proves unfeasible or unreliable. Given this limitation, we perform manual sequencing of the mass spectra.In this study, the bottom-up approach was combined with intact protein analysis for the structural characterization of ependymins from the marine sponges Haliclona caerulea, Haliclona implexiformis, Callyspongia vaginalis, Niphates erecta, and Tedania ignis. With this approach, we were able to fully or partially sequence the proteins under investigation, determine the presence of glycosylation, and identify their interaction with the pigment astaxanthin (M+H⁺ = 597 Da). Our results indicate that sponge ependymins retain structural features of this protein family found in mammals, and that ependymins are carotenoid-binding proteins that specifically bind pigments. Furthermore, this work highlights the importance of adaptive approaches and the value of de novo sequencing for proteomic characterization in contexts where bioinformatics resources are limited.

Agradecimentos: I would like to thank the Federal University of Ceará and the funding agencies that support the research carried out.