A new method for sensitive and accurate proteome quantification in neat plasma

Abstract

Blood is the primary source of protein biomarkers because it is readily accessible and reflects both physiological and pathological changes. However, ion suppression by high-abundance plasma proteins has long limited the coverage of mass spectrometry (MS)-based proteomics. Recent advances in nanomaterial-based enrichment have expanded blood proteome coverage to more than 6000 proteins. Nevertheless, whether the quantities of the enriched proteins proportionally reflect their true levels in neat blood – and thereby faithfully represent disease-associated variation – remains debated. We therefore developed NCTPro (Nanomaterial-enhanced Cross-Channel Transfer strategy for neat blood Proteomics), which enables in-depth plasma proteome quantification without enrichment-induced perturbation of endogenous protein concentrations. Using non-isobaric mass tags, NCTPro combines a reference channel of enriched low-abundance plasma proteins with a neat plasma channel in a single data-independent acquisition mass spectrometry (DIA-MS) run. Deep proteome coverage in the reference channel is transferred to the neat plasma channel through MS1 feature matching and MS2-assisted elucidation. In this way, NCTPro identifies more than 3000 proteins in neat plasma using a 23-min elution gradient – at least a three-fold increase in coverage over recent reports at similar throughput. Spike-in experiments further demonstrate that NCTPro provides accurate fold-change quantification, high precision, and high linearity, making it well suited to a wide range of clinical plasma proteomics studies.