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Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

ABSTRACT: Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method and to compare it with two commercial kits, including HSA and IgG immunodepletion (IMDP) and combinatorial peptide ligand libraries (CPLL), using untreated plasma as a control condition. By varying the temperature and the incubation period, the optimal condition was found as treatment at 95°C for 20 min, which maintained about 1% recovery yield of soluble proteins. Consistency and reproducibility of thermal treatment-derived plasma subproteome were checked by two-dimensional electrophoresis. The coefficient of variation regarding protein spot numbers was less than 10% among three independent specimens. Highly abundant protein depletion of the thermal treatment was evaluated by immunoblotting against HSA and IgG as compared to the untreated plasma, IMDP, and CPLL. Multidimensional comparison based on 489 unique peptides derived from the label-free quantitative mass spectrometry revealed that the thermal treatment, IMDP, and CPLL provided distinct sets of plasma subproteome compared to untreated plasma, and these appeared to be complementary to each other. Comparing the characteristics of the three procedures suggested that thermal treatment was more cost-effective and less time-consuming than IMDP and CPLL. This study proposes the use of thermal treatment as a reliable and cost-effective method for plasma prefractionation which provides benefits to large-scale proteomic projects and biomarker studies.

SUBMITTER: Chiangjong W

PROVIDER: S-EPMC6515177 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

Chiangjong Wararat W Changtong Channarong C Panachan Jirawan J Weeraphan Churat C Srisomsap Chantragan C Hongeng Suradej S Svasti Jisnuson J Chutipongtanate Somchai S

BioMed research international 20190430

Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method a ...[more]

PMID: 31183377

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Project description:Investigation of gene expression in cultured human skin epithelial keratinocytes (HaCaT) following non-thermal plasma treatment for 60 s, compared to untreated control. Non-thermal atmospheric pressure plasma has recently gained attention in the field of biomedical and clinical applications. In the area of plasma medicine research one promising approach is to promote wound healing by stimulation of cells involved. To understand basic molecular and cellular mechanisms triggered by plasma treatment we investigated biological effects of an argon plasma jet (kinpen) on human epithelial skin cells. Consequently, whole-genome microarrays were used to analyze this interaction in detail and identified a statistically significant modification of 3,274 genes including 1,828 up- and 1,446 down-regulated genes. Particularly, plasma-treated cells are characterized by differential expression of a considerable number of genes involved in the response to stress. In this regard, we found a plasma-dependent regulation of oxidative stress answer and increased expression of enzymes of the antioxidative defense system (e.g. 91 oxidoreductases). Our results demonstrate that plasma induces cell reactions of stress-sensing but also of proliferative nature. Consistent with gene expression changes as well as Ingenuity Pathway Analysis prediction, we propose that stimulating doses of plasma may protect epithelial skin cells in wound healing by promoting proliferation and differentiation. In conclusion, gene expression profiling may become an important tool in identifying plasma-related changes of gene expression. Our results underline the enormous clinical potential of plasma as a biomedical tool for stimulation of epithelial skin cells We investigated biological effects of an argon plasma jet on HaCaTs. Microarray were used to analyzed this interaction in detail. The transcripts analyzed in this study are further described in Schmidt et al. (2013): Non-thermal plasma treatment is associated with changes in transcriptome of human epithelial skin cells. Accepted in Journal Free Radical Research

Dataset Information

Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

Publications

Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

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