Jarvinen, E.; Liu, X.; Varjosalo, M.; Keskitalo, S. E.

Plasma is an ideal material for proteomics due to its diverse protein content reflecting physiological and pathological states, and its compatibility with minimally invasive sampling. Deep proteomic profiling of plasma is hindered by the dominance of high-abundant proteins that mask the detection of low-abundant proteins. To overcome this, we compared five plasma protein enrichment methods, MagNet, ENRICHplus, ENRICHiST, EasySep, and EXONET, against neat plasma using LC-MS proteomics. All five methods substantially increased protein identifications, with MagNet, ENRICHplus, EasySep, and EXONET yielding up to 4200 proteins per sample, over 7-fold more than neat plasma, using a 44-minute gradient on the Evosep One and data-independent acquisition on the timsTOF Pro 2. These methods enriched extracellular vesicle-associated proteins while effectively depleting high-abundant proteins. We further optimized the cost-effective MagNet protocol by increasing the plasma-to-bead ratio and automated the workflow, including Evotip loading, on the Biomek i5 liquid handler. Automated MagNet demonstrated high reproducibility and a remarkably low total cost of just a few dollars per sample. This streamlined enrichment strategy enables scalable, high-throughput LC-MS plasma proteomics, supporting biomarker discovery across large clinical cohorts.