Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

Abstract

Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

Figure 1

(a) The iTRAQ-based quantitative platform used for plasma proteome analyses in which the flow-through fractions from four crude plasma samples (A–D) are modified with iTRAQ 4-plex reagents, pooled into a single mixture, and separated with offline SCX-LC-MS/MS. Example chromatograms (λ _280 nm) from three independent injections of plasma sample C upon (b) MD and (c) TMD are shown.

Figure 2

(a) Venn diagram for proteins identified in the human plasma proteome project (HPPP) and the TMD workflow presented herein. (b) Venn diagram for proteins identified in three workflow replicate (WR) experiments.

Figure 3

(a) The distribution of CV values for I ₁₁₅/I ₁₁₄ (grey rectangular), I ₁₁₆/I ₁₁₄ (shaded rectangular), and I ₁₁₇/I ₁₁₄ (black rectangular) for proteins quantified in each of the three independent experiments (N = 139 proteins). The cumulative frequency of proteins with specific CV values for I ₁₁₅/I ₁₁₄ (dashed square), I ₁₁₆/I ₁₁₄ (dashed triangle), and I ₁₁₇/I ₁₁₄ (dashed circle) are shown. CV values are given as fractional values. The total peak intensity is represented by I. (b) Plot of the mean CV values for reporter ion ratios relative to reference channel 114 as a function of the number of spectral counts identified for each protein. Only proteins identified in all three workflow replicates are represented in this plot. (c) Power analysis for iTRAQ-based quantitative platform whereby fold change values are plotted for a given number of biological replicates as a function of the number of technical replicates (i.e., m = 1 to 4). The power and significance level values were set to 80% and 0.05, respectively.