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.