Application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers

Abstract

Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection.The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5days) ranged between 3.2–10.4% and 3.4–12.6%, respectively. In addition, after 22weeks of storage of the Mitra microsampling devices at −80°C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n=3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R=0.8383).In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.