Optimized Hemoglobin Depletion Improves Clinical Proteomics from Dried Blood Spots

Abstract

Abstract Equitable access to large sample cohorts for robust, high-throughput proteomics for biomarker discovery is a major barrier to widescale clinical implementation. Dried blood spots (DBS) offer a minimally invasive alternative to venous blood draws, enabling at-home microsampling (<50 μL) for centralized analysis, thus enhancing research participation. This approach is particularly relevant for under-represented groups, including children, the elderly, minority backgrounds and those with long-term health conditions such as chronic kidney disease (CKD), where disease fluctuations may occur outside the clinic, and vein preservation is critical. Proteomic analysis has demonstrated great utility in monitoring disease progression, and for biomarker/therapeutic target discovery. However, liquid chromatography–tandem mass spectrometry (LC–MS/MS) of whole blood is hindered by the wide dynamic range and the relatively high-abundance of proteins such as hemoglobin, compromising biomarker discovery. Here, we establish an optimized workflow for protein extraction and hemoglobin depletion from microsamples obtained using DBS, enabling sensitive and high-throughput proteomic analysis. We demonstrate that hemoglobin depletion increases protein identifications by ∼50%, mitigating ion suppression and dynamic range effects, enabling the identification of putative biomarkers from patients with stage 5 CKD on dialysis. We also evaluated a commercial cell-free DBS device which yielded a sample more representative of plasma compared to traditional DBS and enabled greater depletion of hemoglobin compared to traditional DBS with hemoglobin depletion methods. Our findings offer a scalable approach for biomarker discovery, facilitating remote, longitudinal clinical studies.