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

Wararat Chiangjong,Jisnuson Svasti,Channarong Changtong,Chantragan Srisomsap,Jirawan Panachan,Churat Weeraphan,Somchai Chutipongtanate,Suradej Hongeng

doi:10.1155/2019/8646039

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.

Highlights

Plasma is an important biological sample for clinical investigations and biomedical research
Since the most abundant protein, human serum albumin (HSA), constitutes over half of the proteins in plasma and can be detected as a 69-kDa protein band on SDS-PAGE, the optimal conditions for thermal treatment were screened by HSA depletion
For the varied temperature-fixed incubation time conditions (65 to 95∘C; 20 min), the prominent band of HSA was markedly decreased at 95∘C thermal treatment comparing to the other lower temperatures (Figure 1(a), left panel)

Summary

Introduction

Plasma is an important biological sample for clinical investigations and biomedical research. Plasma is relatively easy to access and can show significant changes in biological markers, which often relate to pathological conditions. The broad dynamic range of plasma proteins (>10 orders of magnitude) and the overwhelming presence of high abundant proteins human serum albumin (HSA) and immunoglobulin G (IgG), which constitute more than. 60-70% of whole plasma proteins, represent challenges for plasma proteomics [1, 2]. Plasma prefractionation is a prerequisite step to reduce the plasma protein complexity and increase the chance of discovering clinical-relevant biomarkers. General approaches in plasma prefractionation include immunodepletion, affinity enrichment, and fractionation [3]. Choosing the proper prefractionation method can improve the outcome of plasma proteomic projects [3]

Objectives

Methods

Results