Evaluation of volumetric absorptive microsampling and mass spectrometry data-independent acquisition in hemoglobin related clinical markers

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.