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

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

Translational Proteomic Approach for Cholangiocarcinoma Biomarker Discovery, Validation, and Multiplex Assay Development: A Pilot Study

Cholangiocarcinoma (CCA) is a highly lethal disease because most patients are asymptomatic until they progress to advanced stages. Current CCA diagnosis relies on clinical imaging tests and tissue biopsy, while specific CCA biomarkers are still lacking. This study employed a translational proteomic approach for the discovery, validation, and development of a multiplex CCA biomarker assay. In the discovery phase, label-free proteomic quantitation was performed on nine pooled plasma specimens derived from nine CCA patients, nine disease controls (DC), and nine normal individuals. Seven proteins (S100A9, AACT, AFM, and TAOK3 from proteomic analysis, and NGAL, PSMA3, and AMBP from previous literature) were selected as the biomarker candidates. In the validation phase, enzyme-linked immunosorbent assays (ELISAs) were applied to measure the plasma levels of the seven candidate proteins from 63 participants: 26 CCA patients, 17 DC, and 20 normal individuals. Four proteins, S100A9, AACT, NGAL, and PSMA3, were significantly increased in the CCA group. To generate the multiplex biomarker assays, nine machine learning models were trained on the plasma dynamics of all seven candidates (All-7 panel) or the four significant markers (Sig-4 panel) from 45 of the 63 participants (70%). The best-performing models were tested on the unseen values from the remaining 18 (30%) of the 63 participants. Very strong predictive performances for CCA diagnosis were obtained from the All-7 panel using a support vector machine with linear classification (AUC = 0.96; 95% CI 0.88–1.00) and the Sig-4 panel using partial least square analysis (AUC = 0.94; 95% CI 0.82–1.00). This study supports the use of the composite plasma biomarkers measured by clinically compatible ELISAs coupled with machine learning models to identify individuals at risk of CCA. The All-7 and Sig-4 assays for CCA diagnosis should be further validated in an independent prospective blinded clinical study