Automated sample preparation for in-depth plasma proteomics

The blood plasma proteome is a promising source of biomarkers, offering the advantage of minimal invasive collection and widespread clinical availability. However, sample-heterogeneity and wide dynamic range of protein abundances hinders comprehensive profiling within the necessary throughput for large cohort studies. To address this challenge, we present a fully automated sample preparation on the Fluent® applying the standardized ENRICH-iST technology that facilitates the identification and quantification of low abundant proteins from plasma. Combined with the next-generation dia-PASEF® MS acquisition, it is designed for high-throughput yet profound characterization of the plasma proteome of large cohorts. The automation platform consists of the Fluent® liquid-handling system (Tecan) with an integrated positive pressure and evaporator module, enabling automation from sample to dried peptides. The automated sample processing covers lysis, enrichment of low abundant proteins, tryptic digestion and clean-up including peptide drying by evaporation for LC-MS ready peptides employing ENRICH-iST technology (PreOmics). A total of 96 samples/run can be prepared for LC-MS analysis within one working day processing 20 µL of blood plasma/ sample. For mass spectrometry-based analysis 300 ng of peptides were separated on the nanoElute® 2 system and analyzed using the dia-PASEF® acquisition program on the timsTOF HT (Bruker). The data were searched with Spectronaut using directDIA+ (Biognosys AG). The high-throughput proteomic plasma preparation underwent a comprehensive evaluation, comprised of a comparative analysis between the automated and manual ENRICH-iST workflow, processing plasma samples of three healthy donors. Remarkably, identification rates and quantitative performance of the automated workflow were found to be equivalent to those obtained through the manual process. When comparing against neat plasma preparation, both manual and automated workflow demonstrated efficient dynamic range compression, yielding an approximate two-fold increase in identifications with application of ENRICH-iST over neat plasma preparation. To further assess the robustness of the automated pipeline, an inter-plate comparison spanning three days was conducted. The Inter-Day study revealed low variability in protein group identifications and their abundance across independent sample preparation runs, affirming the reliability of the automated plasma pipeline. The workflow flexibility, accommodating samples sizes ranging from 1 to 96, enables seamless processing of both small- and large-scale cohorts. This adaptability was exemplified by processing an age-dependent differences, yielding high-quality data to provide valuable biological insights across diverse experimental settings. This automated plasma proteome pipeline combines scalable throughput with profound proteomic characterization, bringing simplicity into the field of plasma proteomics.