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

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%. 1. Introduction Discovery studies using plasma proteomics present challenges due to the technical difficulties associated with measuring the large dynamic range (~10–12 orders of magnitude) of proteins that exist in this medium [1]. Low-abundance proteins, which are of interest for biomarker applications, are often only accessible with involved proteomics workflows that utilize multiple sample fractionation steps. While the development of specific clinical immunoassays would resolve this approach, much work needs to be done in this area. Enrichment strategies for low-abundance plasma proteins rely on immunodepletion of high-abundance proteins [2–5], and, more recently, tandem depletion strategies have been employed [6–9]. For example, proteins present in as little as 1–1.6？μg·mL？1 concentrations are detectable using tandem removal of abundant proteins with human serum albumin and Human 14 (Hu 14) multiple affinity removal system (MARS) columns [9]. A two-stage depletion setup that involves serial IgY and Supermix columns has also been effective in increasing the number of detectable low abundance proteins without affecting quantitative accuracy and precision using isobaric tags for relative and absolute quantification (iTRAQ) [6]. Recently, an updated reference