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Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion.

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.

SUBMITTER: Liu Z

PROVIDER: S-EPMC5104378 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion.

Liu Zhao Z Fan Songhua S Liu Haipeng H Yu Jia J Qiao Rui R Zhou Mi M Yang Yongtao Y Zhou Jian J Xie Peng P

PloS one 20161110 11

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. I ...[more]

PMID: 27832179

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Project description:Graphical abstract Highlights • A rapid, sensitive, and specific flow cytometry assay was developed to detect anti-PEG IgG and IgM in human blood plasma.• Using the method, anti-PEG IgG or IgM were detected in 65% of plasma samples from 300 healthy blood donors.• The presence of anti-PEG IgG and IgM was confirmed using three validation assays.• The highest prevalence of both anti-IgG and anti-IgM was in individuals 18–24 years of age.• No correlation was found between anti-PEG IgG and IgM concentrations. Polyethylene glycol (PEG) is a biocompatible polymer used in biotherapeutics to increase bioavailability, reduce the frequency of administration, and optimize pharmacokinetics. Anti-PEG antibodies have been detected in healthy individuals and may decrease efficacy and alter the pharmacokinetics of PEGylated therapeutics; however, the prevalence of anti-PEG antibodies is unclear. In this study, a flow cytometry assay was optimized to detect anti-PEG IgG and IgM in human blood plasma. Three hundred (300) plasma samples from healthy blood donors were screened; anti-PEG IgG or IgM was detected in 65.3% of the total population, with 21.3% having anti-PEG IgG, 19.0% having anti-PEG IgM, and 25.0% having both anti-PEG IgG and IgM. The presence of anti-PEG IgG and IgM was confirmed using a 0.5% Tween-20 interference assay, a 20 kDa PEGylated polystyrene bead binding assay, and Western blotting of purified plasma from human IgG and IgM purification columns. The concentrations of anti-PEG IgG and IgM in positive samples ranged from 39 ng/mL to 18.7 ?g/mL and 26 ng/mL to 11.6 ?g/mL, respectively. The highest prevalence of both anti-IgG and anti-IgM was in individuals 18–24 years of age. The prevalence of anti-PEG IgG and IgM tended to be higher in women but did not differ among races. Age, sex, and race were not associated with the concentrations of anti-PEG IgG or IgM. No correlation was found between anti-PEG IgG and IgM concentrations. Our study indicates that flow cytometry can be used to detect anti-PEG IgG and IgM antibodies in human plasma.

Dataset Information

Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion.

Publications

Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion.

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