Maria Gabriela Vieira Oliveira da Silva^1,2, Juliana Hilda de Albuquerque Gomes^1,2, Maria Helena Menezes Estevam Alves², Roberto Afonso da Silva², Danyelly Bruneska Gondim Martins^1,2

Proteomic profiling offers a robust approach for elucidating molecular patterns in patient cohorts. By leveraging protein-protein interaction mapping, ontological and metabolic analyses, it is possible to identify specific biomarkers for various pathologies. This study conducted a proteomic analysis of plasma samples from patients in Recife using a quadrupole time-of-flight (QToF) mass spectrometer with ion mobility-enabled. The objective was to evaluate protein behavior using two distinct methodologies: 2D 5-fractions dilution (2DD) and single pump trap (SPT). Protein detection and identification were performed using a high-resolution mass spectrometer coupled with NanoUPLC (SYNAPT XS). This instrument is capable of processing samples via two dimensions low-flow (2DD) or continuous flow (SPT) method, which corresponds to a more complex separation (5 fractions) and a longer and continuous gradient, respectively. A total of 36 plasma samples were used, categorized into four groups: Group 1 - Positive Control (COVID-19 and Immune-mediated Disease - IMD) (6 patients); Group 2 - IMD only (11 patients); Group 3 - COVID-19 only (8 patients); and Group 4 - Negative Control (11 individuals). Bioinformatic tools such as Python, R, OmicScope, VolcaNoseR, MetaScape and String were employed to characterize and interpret protein functions and signaling pathways. In the 2DD analysis, 293 proteins were detected, of which 141 were differentially expressed proteins (DEPs). The SPT technique identified 175 proteins, with 104 being DEPs. A cross-analysis between the groups, using both spectrometer injection techniques, revealed a similar number of proteins, though with limited reproducibility, at approximately 20–25%. Gene ontology analysis indicated that, for both methods, key biological processes included the humoral immune response, complement and coagulation cascades, hemostasis, platelet degranulation, and the SARS-CoV-2 signaling pathway network. The 2DD technique also highlighted the involvement of neutrophil degranulation and the acute inflammatory response, while the SPT technique additionally revealed processes such as the negative regulation of leukocyte activation, adaptive immune response, cholesterol metabolism, thyroid hormone synthesis, regulation of proteolysis, and the Wnt signaling pathway. Despite the observed difference in the number of identified proteins, no significant variations were found in the enrichment and ontology analyses between the two techniques. These findings, however, raise a critical question for biomarker discovery: "Which proteomic injection technique—two dimensions low-flow (2DD) or continuous (SPT)—is more effective for reliably identifying disease-related protein biomarkers from blood samples?"

Agradecimentos: We would like to thank the CAPES, CNPq and the Keizo Asami Institute (iLIKA) for all their support in contributing to the development of this work.