Data-independent acquisition (DIA) allows comprehensive proteome coverage, and it has the potential to work as a unified protocol to determine a multitude of proteins in blood. Because of its high specificity, mass spectrometry has a high potential to reduce the interferences presented by other assays in the evaluation of blood markers. Here, we combined DIA with volumetric absorptive microsampling (VAMS) and automated proteomics sample processing as a platform to perform clinical markers determination. We evaluate, as a proof-of-concept, two hemoglobin-related clinical biomarkers: glycated hemoglobin (HbA1c) and hemoglobin variants. HbA1c by DIA showed good correlation with the reference method, but method imprecision did not meet the very low imprecision specification for the clinical evaluation of this biomarker. A strategy for identification of Hb variants was developed based on a customized database combined with a workflow for DIA data extraction and rigorous evaluation of peptides. This approach revealed Hb variants not identified by routinely clinical methods such as Hb Woodville and Hb Okayama.

DIA experiments were performed alternating one full scan from m/z 360 to 900 at 60,000 mass resolution followed by 16 MS/MS scans with 25 m/z precursor isolation windows, at 15,000 resolution, AGC target 5e5, maxIT 55 ms with NCE of 30%. Isolation windows placements were optimized by Skyline and covered from m/z 350 to 800 using an overlapping window of 0.5 m/z.

Thirty-six blood specimens previously evaluated for hemoglobinopathies were included in this study. The routine protocol for hemoglobinopathies characterization was based on capillary zone electrophoresis (CZE) followed by cation exchange high performance liquid chromatography (CEX-HPLC). CZM was achieved in Capillarys 2 Flex Piercing System (Sebia, Lisses, France) which includes automated processing for hemolysis, separation based on electrophoretic mobility and spectrophotometric detection at 415 nm. The identification of variants within defined zones was obtained using software Phoresis (version 9.3.0). CEX-HPLC was performed in Variant II Hemoglobin Testing System (Bio-Rad, Hercules, CA, USA) equipped with β-Thalassemia Short Program kit and controlled by CDM 5.1.1software.