Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach

The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary exosome-enriched extracellular vesicles (EVs). We developed a SWATH-MS workflow with a curated spectral library of 1,073 targets. Application of the workflow across nine replicates of three sample types (EVs, microvesicles (MVs) and urine proteins (UP)) resulting in the quantitation of 842 proteins. The median-coefficient of variation of the 842 proteins in the EV sample was 7.6%, indicating excellent reproducibility. Data analysis showed common EV markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary EVs as compared to MV and UP samples. Further development and applicationof this SWATH-MS workflow to a variety of kidney diseases may allow for new and robust avenues for biomarker identification and validation for clinical use.