Uncovering the Plasma Proteome: A Comparative Evaluation of Immunoaffinity Depletion and Nanoparticle-Based Enrichment for Mass-Spectrometry-based Plasma Proteomics

Blood plasma is rich in protein biomarkers that may indicate disease state. However, 95% of the plasma proteome consists of high-abundance proteins (HAPs), like immunoglobulins and albumins, which mask low-abundance biomarkers during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This study evaluated two plasma-depletion techniques, the Thermo Scientific High-SelectTM Top 14 Abundant Protein Depletion Columns and Ceres Nanosciences Nanotrap® Protein Enrichment Affinity Kits, to compare reproducibility and proteomic coverage in biomarker detection. Initial analysis used an Orbitrap Exploris 480 mass spectrometer coupled to an EASY-nLC nanoLC system with a 90-minute gradient, and data was processed using MSFragger with a 5% false discovery rate (FDR) threshold. For verification, samples were analyzed using a Bruker trapped ion mobility spectrometry–time-of-flight (timsTOF) mass spectrometer and processed using Spectronaut at a 1% FDR. The timsTOF platform identified a similar number of proteins, despite analyzing fivefold-diluted samples and using a shorter 20-minute LC gradient. The High-Select depletion columns identified more unique proteins than Nanotrap enrichment. However, both methods yielded similar distributions of protein families and comparable proportions of glycosylated and phosphorylated proteins, suggesting that the two approaches captured similar functional and post-translational proteome profiles. Differences were observed in the subcellular origin of the identified proteins. High-Select–processed samples exhibited a nearly equal distribution of extracellular (50.37%) and intracellular (49.63%) proteins, whereas Nanotrap-enriched samples were predominantly extracellular (68.82%). This finding suggests that High-Select depletion may provide greater recovery of intracellular proteins present in plasma, including proteins released through cellular turnover, tissue injury, or cell lysis.