Comparative analysis of plasma affinity depletion methods: Impact on protein composition and phosphopeptide abundance in human plasma

Abstract Affinity‐based protein depletion and TiO 2 enrichment methods play a crucial role in detection of low‐abundant proteins and phosphopeptides enrichment, respectively. Here, we assessed the effectiveness of HSA/IgG (HU2) and Human 7 (HU7) depletion methods and their impact on phosphopeptides coverage through comparative proteome analysis, utilizing in‐solution digestion and nano‐LC‐Orbitrap mass spectrometry (MS). Our results demonstrated that both HU2 and HU7 affinity depletion significantly decreased high‐abundant proteins by 1.5–7.8‐fold ( p < 0.001). A total of 1491 proteins were identified, with 48 proteins showing significant expression in the depleted groups. Notably, cadherin‐13, neutrophil defensin 1, APM1, and desmoplakin variant protein were exclusively detected in the HU2/HU7‐depleted groups. Furthermore, study on effect of depletion on phosphopeptides revealed an increase in tandem MS spectral counts with notable decrease (∼50%) in peptide spectrum matching in depleted groups, which was attributed to significant reduction in protein counts. Our post translation modification workflow for phosphoproteomics detected 42 phosphorylated peptides, corresponding to 12 phosphoproteins with unique peptide match ≥2 (high false discovery rates confidence). Among them, 10 phosphorylated proteins are highly expressed in depleted groups. Overall, these findings offer valuable insights in selection of protein depletion methods for comprehensive plasma proteomics analysis.