Background: Blood plasma represents a valuable reservoir of disease biomarkers, but the discovery of potential protein biomarkers in human plasma poses significant challenges. The intricacies of the plasma proteome, marked by its high complexity and wide dynamic range of proteins, hinder comprehensive profiling within the necessary throughput for large cohort studies. In response to this challenge, we have devised an automated sample preparation pipeline utilizing the Fluent® liquid handling system, which incorporates the innovative ENRICH-iST plasma workflow (PreOmics). This workflow not only enables the identification and quantification of low-abundance proteins, but is also tailored for the high-throughput, in-depth characterization of the plasma proteome in large study populations.
Methods: Our automated platform integrates the Fluent® automated liquid-handling workstation in conjunction with a newly developed positive pressure module (Tecan). This automated sample processing encompasses every stage of the ENRICH-iST workflow (PreOmics), from enriching low-abundance proteins to preparing peptides ready for LC-MS analysis. Remarkably, this platform can process 96 samples in a single run and one working day, requiring only 20 µL of blood plasma per sample. Peptides (300 ng) were separated with a 20-min gradient on the nanoElute 2 system and analyzed using dia-PASEF® acquisition on the timsTOF HT (Bruker) Spectronaut 17 was used for direct DIA analysis (Biognosys).
Results: Our high-throughput plasma pipeline was rigorously assessed by simultaneously processing of 96 samples sourced from three healthy donors. The entire automated sample preparation process, from raw plasma sample to clean peptides, took approximately five hours and demanded minimal user intervention. Our data analysis revealed that the inclusion of plasma enrichment in the workflow led to a more than twofold increase in protein identifications compared to neat plasma analysis. Furthermore, we observed low technical variability within replicates, underscoring the robustness and reliability of this automated plasma pipeline.
Conclusions: This automated plasma proteome pipeline expertly combines high throughput with comprehensive proteome characterization simplifying plasma proteomics. It promises to enhance our ability to uncover valuable biomarkers in large-scale studies of human plasma.