Optimization of SAX-Based enrichment of plasma extracellular vesicles for high-throughput deep phenotyping of the heart failure syndrome

Introduction

Heart failure syndrome (HF), affecting approximately 15 million Europeans, ranks as the leading cause of hospitalization. HF profoundly diminishes quality of life and life expectancy, imposing a substantial burden on public health. Treating patients with HF remains a significant challenge due to limited understanding of a pathophysiology of the heterogeneous HF syndrome, limited effectiveness of existing prevention strategies and its progressive nature. The aim of the study is to develop high throughput, DIA- based, deep coverage plasma proteomics workflow for screening of patients, to suggest novel hypotheses about the molecular mechanisms of HF syndrome that explain how the identified biomarkers contribute to the disease.

Methods:

We established a SAX-bead based protocol for enrichment of plasma extracellular vesicles in 96-well plate format using a Biomek i7 liquid handling station to enable digestion throughput of 450 samples per week. Initial method evaluation was performed on smaller cohort of 144 diabetes patients treated with empagliflozin vs. placebo, examined three times during the phase of 12 weeks. We evaluated DIA- based acquisition workflows on Orbitrap Exploris 480 coupled to Ultimate 3000 and timsTOF Pro2 coupled to nanoElute2, aiming to provide deeper knowledge about proteomics signatures associated with HF. The data were processed DIA-NN using both spectral library and library-free approaches.

Results and conclusion:

Label-free DIA analysis of the plasma samples using 30 min method (50 SPD) resulted in the quantification of 1282 unique protein groups (n=2) in Orbitrap and 1269 in timsTOF Pro2 in the library-free approach with the overlap of 92% between two platforms. Our established method shows excellent reproducibility with the median CV < 15% and Pearson correlation of 0.98 between the digestion QCs. Using a gas-phase fractionation generated spectral library in the timsTOF Pro 2 platform increased the number of quantified unique proteins to 1631. The SAX -beads based automated protocol has been demonstrated to ensure deep coverage of plasma proteome, but also to be reproducible, cost-effective and usable in high-throughput for clinical applications.