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Patel BB , Barrero CA , Braverman A , Kim PD , Jones KA , Chen DE , Bowler RP , Merali S , Kelsen SG , Yeung AT
Assessment of Two Immunodepletion Methods: Off-Target Effects and Variations in Immunodepletion Efficiency May Confound Plasma Proteomics
Journal of Proteome Research. 2012 Dec;11(12) :5947-5958
PMID: WOS:000311925900034 PMCID: PMC 3518753
AbstractImmunodepletion 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 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.
NotesPatel, Bhavinkumar B. Barrero, Carlos A. Braverman, Alan Kim, Phillip D. Jones, Kelly A. Chen, Dian Er Bowler, Russell P. Merali, Salim Kelsen, Steven G. Yeung, Anthony T. National Institutes of Health (NIH) [RC2 HL101713]; National Cancer Institute [N01-CN-43309]; Driskill Foundation [P30CA06927]; Commonwealth of Pennsylvania; Pew Charitable Trust; Kresge Foundation We thank Brian Searle for critical reading of this manuscript and Dr. Karen Kawarta, Sigma-Aldrich, for performing the immunodepletion procedures. This work was partially supported by the National Institutes of Health (NIH) Grant RC2 HL101713, the National Cancer Institute, Work Assignment #16 of N01-CN-43309, the Driskill Foundation, Institutional Core Grant P30CA06927, Tobacco Settlement Funds from the Commonwealth of Pennsylvania, the Pew Charitable Trust, and the Kresge Foundation. 28 Amer chemical soc Washington 048pv