Quantitative screening of serum protein biomarkers by reverse phase protein arrays.

Zhizhou Kuang,Yu-Hua Yi,Ruochun Huang,Zhiqiang Lv,Fuping Xu,Ruo-Pan Huang,Jian Wu,Zhimin Yang,Xinyan Chen

doi:10.18632/oncotarget.25976

Abstract

Screening biomarkers in serum samples for different diseases has always been of great interest because it presents an early, reliable, and, most importantly, noninvasive means of diagnosis and prognosis. Reverse phase protein arrays (RPPAs) are a high-throughput platform that can measure single or limited sets of proteins from thousands of patients' samples in parallel. They have been widely used for detection of signaling molecules involved in diseases, especially cancers, and related regulation pathways in cell lysates. However, this approach has been difficult to adapt to serum samples. Previously, we developed a sensitive method called the enhanced protein array to quantitatively measure serum protein levels from large numbers of patient samples. Here, we further refine the technology on several fronts: 1. simplifying the experimental procedure; 2. optimizing multiple parameters to make the assay more robust, including the support matrix, signal reporting method, background control, and antibody validation; and 3. establishing a method for more accurate quantification. Using this technology, we quantitatively measured the expression levels of 10 proteins: alpha-fetoprotein (AFP), beta 2 microglobulin (B2M), Carcinoma Antigen 15-3(CA15-3), Carcinoembryonic antigen (CEA), golgi protein 73 (GP73), Growth differentiation factor 15 (GDF15), Human Epididymis Protein 4 (HE4), Insulin Like Growth Factor Binding Protein 2 (IGFBP2), osteopontin (OPN) and Beta-type platelet-derived growth factor receptor (PDGFRB) from serum samples of 132 hepatocellular carcinoma (HCC) patients and 78 healthy volunteers. We found that 6 protein expression levels are significantly increased in HCC patients. Statistical and bioinformatical analysis has revealed decent accuracy rates of individual proteins, ranging from 0.617 (B2M) to 0.908 (AFP) as diagnostic biomarkers to distinguish HCC from healthy controls. The combination of these 6 proteins as a specific HCC signature yielded a higher accuracy of 0.923 using linear discriminant analysis (LDA), logistic regression (LR), random forest (RF) and support vector machine (SVM) predictive model analyses. Our work reveals promise for using reverse phase protein arrays for biomarker discovery and validation in serum samples.

Highlights

Biomarkers are key indicators used for early, rapid and accurate diagnosis for most diseases
The intra-assay and inter-assay CV of the 7 glass slide assays were significantly higher, ranging 21.37–40.19% and 16.33–131.47%, respectively for the 6 serum samples. These results suggest that the NC membrane has advantages as a solid support matrix in stability and repeatability for Reverse phase protein arrays (RPPAs) when assaying serum samples
In RPPA, thousands of individual samples are immobilized on a solid support matrix by an arrayer so that the arrayed samples can be recognized simultaneously with highly specific antibodies against desired targets

Summary

Introduction

Biomarkers are key indicators used for early, rapid and accurate diagnosis for most diseases. For identification of protein biomarkers, highthroughput screening platforms have shown powerful technical advantages, differing from traditional approaches such as ELISA and western blot. Two technologies including antibody arrays and reverse phase protein arrays (RPPAs) have been wildly employed in the discovery and validation of biomarkers and have produced promising results for many diseases including cancers, cardiovascular diseases and neurodegeneration diseases [7]. RPPA is a cost–effective and robust platform offering a high-throughput approach to screen biomarkers or validate candidate markers with a tiny amount of sample over a huge population of samples. This is ideal for projects requiring observation over time, before and after treatment, between disease and non-disease states as well as between responders and nonresponders, etc. Since the first publication in 2001 [8], RPPAs have been successfully applied in monitoring epigenetic changes of proteins, such as phosphorylation, involved in disease-related regulation pathways in tissue or cell lysate samples, especially in cancers [9,10]

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Conclusion