Lorena Vitória da Costa Sousa¹, Jair Mendes Lima Ferreira Junior¹, Bárbara Cibelle Soares Farias Quintela¹, Anna Carolina Machado Marinho¹

Introduction: Proteomics is the large-scale study of proteins, their functions, and structures, improving our understanding of biological processes and diseases. Proteomic analysis of plasma by mass spectrometry can identify biomarkers that signal various health conditions, and can be used for early disease diagnosis, prognosis, monitoring, and provide insights into underlying mechanisms. Objective: To compare two methods for depleting highly abundant proteins in human plasma: acid precipitation/organic solvent (method 1) and a commercial kit (method 2). Materials and methods: For method 1, raw plasma was diluted, and proteins were precipitated using acetic acid and acetonitrile (ACN). For method 2, 10 µL of crude plasma was depleted with Cibracon blue and Protein A gel, following the manufacturer\'s guidelines, desalted, and buffer changed to ammonium bicarbonate. Both samples were resolved by SDS-PAGE, reduced and alkylated, then incubated at 37 °C for 16 hours. The samples were injected and analyzed by Orbitrap mass spectrometry (Q Exactive Plus), with fragmentation of the most abundant precursor ions. The mass spectra were compared with the human proteome in UniProt KnowledgeBase, using PatternLab software for data analysis. Results and discussion: Gel analysis revealed lower protein diversity in acid/ACN-depleted samples compared to those treated with the commercial kit. Albumin was still detected, albeit at lower levels, suggesting incomplete depletion even with the commercial kit. MS analysis detected 70 proteins in the sample depleted by method 1 and 114 proteins in the sample depleted by method 2, with 54 proteins unique to method 2. The five most abundant proteins were: alpha-1 acid glycoprotein, immunoglobulins, haptoglobin, serotransferrin, and apolipoprotein A-I. Conclusion: Depletion with a commercial kit was more effective than acid precipitation/organic solvent. However, more efficient commercial depletion kits are needed to better remove abundant proteins, enabling improved MS detection and quantification of low-abundance proteins, which are essential for identifying key proteins in some diseases.

Agradecimentos: Fiocruz , SUS, PROVOC, EEEP de Queiroz, Escola Politécnica Joaquim Jose Venancio