Depletion of abundant plasma proteins and limitations of plasma proteomics

Abstract

Immunoaffinity depletion with antibodies to the top 7 or top 14 high-abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low-abundance (<10 ng mL(-1)) plasma proteins were detected, they accounted for only 5-6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms-even with immunodepletion-cannot be expected to efficiently discover low-abundance, disease-specific biomarkers in plasma.

Figure 1

Linear correlations of the spectral counts between pairs LC-MS/MS analyses of three replicate MARS-14 FT fractions. Spearman correlation coefficient (r) is shown for each comparison. Similar results were obtained with MARS-7 depleted flow-through samples.

Figure 2

Nontargeted human plasma proteins that bind to the MARS-7 and MARS-14 column in replicate LC-MS/MS analyses. The plotted values represent the fraction of the total counts for each protein that remain bound (i.e., B/(B + FT)).

Figure 3

Comparison of proteome inventories from crude plasma, MARS-7 flow-through and MARS-14 flow-through analyzed by IEF-LC-MS/MS. A single IDpicker report was created from 3 separate single-dimensional MS/MS analyses and filtered for immunoglobulins, contaminant proteins and proteins identified by reverse sequences. A total of 220 proteins detectable by at least 10 spectral counts were included in this comparison.

Figure 4

Linear correlation between the spectral counts for each protein and previously reported concentrations of plasma proteins (Table S3.).

Figure 5

Global impact of MARS-7 (A) and MARS-14 (B) immunodepletion on detection of plasma proteins. Proteins observed with at least 10 spectral counts in the study are represented and are ranked by spectral counts in unfractionated (crude) plasma. Plotted values (y-axis) are the log₂ ratios of detected spectral counts for each protein in depleted plasma relative to counts for the same protein in unfractionated plasma.

Figure 6

The distribution of spectral counts for identified proteins (A and B) and peptides (C and D) in unfractionated plasma (blue line), MARS-7 FT (red line), MARS-14 FT (green line) and RKO cell lysate (purple line). Proteins and peptides are ranked on the x-axis in order of decreasing spectral counts in unfractionated plasma. The y-axis indicates cumulative spectral counts as a function of peptide or protein rank. Data are plotted for the top 50 proteins and the top 100 peptides. Results shown are for a single analysis by either reverse phase LC-MS/MS (A and C) or IEF-LC-MS/MS (B and D).

Figure 7

The distribution of MS1 signal intensities for identified proteins (A and B) and peptides (C and D) in unfractionated plasma (blue line), MARS-7 FT (red line), MARS-14 FT (green line) and RKO cell lysate (purple line). Proteins and peptides are ranked on the x-axis in order of decreasing signal intensity in unfractionated plasma. The y-axis indicates cumulative MS1 signal intensity as a function of peptide or protein rank. Data are plotted for the top 50 proteins and the top 100 peptides. Results shown are for a single analysis by either reverse phase LC-MS/MS (A and C) or IEF-LC-MS/MS (B and D).