Plasma proteome profiling is challenging due to the absence of efficient workflows that (i) allow for a deep dive into the plasma proteome, (ii) utilize existing automation infrastructure, (iii) are scalable in terms of ability to process large patient cohorts in a reproducible, and cost-effective manner. Here we present an evolution of the Mag-Net method for deep plasma profiling (Wu et al, 10.1101/2023.06.10.544439) by employing combinations of hyper-porous magnetic microparticles as well as further optimizing the workflow by integrating additional orthogonal steps for plasma depletion and/or enrichment aimed at further increasing proteome depth.

Plasma from a single donor was collected using EDRN SOP (Tuck et al., 10.1021/pr800545q), a range of hyper-porous beads with varying functional groups, together with MagReSyn® SAX as a control, were utilized with the Mag-Net protocol in different combinations. Further depletion and enrichment steps were integrated with Mag-NetTM. The methods were performed in semi-automated manner using a King Fisher magnetic handling platform or fully automated on an Opentrons OT2 robot. Peptides were loaded onto Evotips and analyzed using an Evosep One coupled to a Bruker timsTOF HT. Data was processed using DIA-NN (version 1.8.1) in library-free mode. Post data processing was performed in R for the comparison of each experimental condition.

Preliminary data indicates that the use of novel mixed mode magnetic beads increases plasma proteome depth by approximately 25%. Whilst the addition of complementary enrichment steps utilizing bead combinations increase plasma proteome coverage by 30%, identifying up to 3,600 protein groups at a throughput 100SPD. This was achieved without sacrificing robustness or the requirement for additional LCMS time. The evaluation of additional bead chemistries and workflow variations are ongoing.