Assessment of Hemoglobin-related clinical markers using volumetric absorptive microsampling and data-independent acquisition mass spectrometry

Data-independent acquisition (DIA) offers extensive proteome coverage and serves as a unified protocol for quantifying numerous blood proteins. With its high specificity, mass spectrometry significantly reduces interference commonly encountered in other blood marker assays. In this study, we integrated DIA with volumetric absorptive microsampling (VAMS) for whole blood collection and storage with automated proteomics sample processing to evaluate clinical markers. As a proof of concept, we assessed two hemoglobin-related biomarkers: glycated hemoglobin (HbA1c) and hemoglobin (Hb) variants. Our results demonstrated that HbA1c quantification via DIA correlated well with the reference method, though the method's imprecision did not meet the required quality standards for this biomarker. Additionally, we developed proposed an approach for hemoglobin variant identification based on a customized hemoglobin database combined with a workflow for DIA data extraction and rigorous evaluation of peptides including multiple qualifiers.