Assessment of Two Immunodepletion Methods: Off-Target Effects and Variations in Immunodepletion Efficiency May Confound Plasma Proteomics

Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin + immunoglobulins and Seppro IgY14 + SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin + immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14 + SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14 + SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.