Abstract
The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest.
Highlights
The discovery of reliable disease biomarkers is one of the biggest concerns for researchers [1,2,3,4,5]
Human plasma was used as a source of proteins, and the whole-plasma proteins were precipitated with using polyethylene glycol (PEG) at various concentrations (4, 8, 12, 16, 20, 24, and 30%)
We found that the numbers of proteins and unique peptides identified using the PEG fractionation (PEGF)-immunoaffinity depletion (IAD) method were increased by 32.5±5.3% and 30.0±3.6% compared with that using the single IAD treatment, respectively, which were mainly reflect in the exclusively identified proteins and unique peptides
Summary
The discovery of reliable disease biomarkers is one of the biggest concerns for researchers [1,2,3,4,5]. It contains various proteins that are actively secreted or presented following cell and tissue leakage [3]. The detection limits of MS imposed by the various ionization processes employed impact both the complexity and dynamic range of analytes that are measurable [13]. These disadvantages are reflected by the predominance of high- and moderate-abundance proteins (HAPs and MAPs), which clearly hampers the identification and quantification of potential low-abundance protein (LAP) biomarkers [2]. During the last few decades, the most commonly used approach to facilitate LAP analysis has been to reduce plasma sample complexities by fractionation [15]
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