Immunoaffinity depletion of high abundance plasma proteins is frequently employed to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. Here we present a detailed evaluation of the Agilent Multiple Affinity Removal SystemTM ( MARS-7 and MARS-14) in the shotgun proteomic analysis of human plasma. We used a multidimensional analysis platform that combines peptide isoelectric focusing (IEF) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze unfractionanted and MARS-7 and MARS-14-depleted plasma proteomes. The MARS colums afforded highly repeatabile and efficient plasma protein depletions and a global enrichment in non-target plasma proteins of 2-4-fold, as assessed by MS/MS spectral counting. Immunodepletion resulted in a 30-40% increase in identified proteins compared to unfractionated plasma. Relatively few non-target proteins were captured by the MARS-7and MARS-14 columns . Although some low abundance (<10 ng mL-1) plasma proteins were detected, they accounted for only 5-6% of total protein identifications in MARS-depleted plasma. Analyses of unfractionated or immunodepleted plasma yielded only 10-20% of the identifications made from identical amounts of the human colon tumor RKO cells. This disparity in proteome detection is due to a much steeper protein abundance distribution in plasma than in cells. These findings demonstrate the consistent performance of abundant protein depletion with MARS columns, but also illustrate the limitations of shotgun proteomics to detect disease biomarkers in plasma proteomes.
Citation: Journal of Proteome Research
Pub Type: Journals
plasma, high-abundance protein depletion, multiple affinity removal system, isoelectric focusing, shotgun proteomics, futility