Leukocyte p53 protein biosignature through standard-aligned two-dimensional immunoblotting

Abstract

Peripheral leukocytes may reflect systemic disease and stress states through their gene expression profile. Subsequent protein analyses of leukocytes are hypothesized to provide essential information regarding systemic diseases. We have developed a protein biosignature analysis of the tumour suppressor and cell stress sensor p53 based on two-dimensional gel electrophoresis and immunoblotting, and utilize fluorescently labelled reference standards to significantly improve the alignment and comparison of biosignatures, including full-length p53 and isoforms p53β and p53γ. Analysis of the p53 biosignatures of peripheral blood mononuclear cells from 526 healthy individuals and 65 acute myeloid leukaemia patients indicated a novel putative p53 protein variant in a subset of individuals (227 of 526 healthy tested). The p53 variant was more distinct in the reference standard aligned biosignatures of healthy individuals, compared to the non-standard aligned leukaemia biosignatures. This approximately 2 kDa heavier variant of p53 appeared with similar frequency in leukemic and healthy test persons, without coinciding with known splice forms or post-translational modifications of p53. We propose that a standardized leukocyte protein biosignature of p53 provides a powerful research tool and indicate how p53 protein biosignatures may be used in future diagnostics.

This article is part of a Special Issue entitled: Integrated omics.

Introduction

Analysis of gene and protein expression in peripheral blood leukocytes (PBL) may provide diagnostic information about healthy and diseased individuals [1], [2]. Significant changes in leukocyte gene expression have been demonstrated in autoimmune diseases [3], [4], [5], Parkinson's disease [6], coronary artery disease and arterial hypertension [7], [8] and Alzheimer's disease [9], [10]. These studies propose standard blood samples as a source of more advanced biomarkers for disease predisposition and early diagnosis than those currently in clinical use [11].

Similarly, the tumour suppressor and cell stress sensor protein p53 has been suggested as biomarker and therapy target in not only cancer but also autoimmune disorders [3], [11]. Leukocyte derived p53 protein is suggested as a biomarker of Alzheimer's disease and as a therapeutic target in rheumatoid arthritis [3], [10], [12]. Leukocyte gene expression profiling of cell death and DNA repair genes such as p53 has been proposed as predictive markers of radiation-induced adverse side effects [13]. Screening of the entire p53 network has also been employed in analysis of individualized cancer susceptibility analysis [11]. The potential of such p53 protein analysis has previously been demonstrated in leukaemia [14], [15], [16], [17], [18], [19], [20], [21], [22].

Alternative splicing of TP53 mRNA and alternative initiation of transcription results in distinct protein isoforms [23], [24]. Analysis of the p53 protein in a biosignature consisting of multiple p53 isoforms may thus be more informative in elucidation of their functions. The twelve identified p53 protein isoforms comprise different combinations of the structural and functional domains of p53 (reviewed in [25], [26]). To detect p53 protein isoform expression in peripheral blood mononuclear cells (PBMCs), we have utilized 2-D gel electrophoresis (2-DE), that identifies small variations in the expression of selected isoforms [15]. 2-DE is a powerful and ubiquitous method for analyzing complex protein mixtures. Subsequent immunoblotting after 2-DE provides information on low-level expression of protein isoforms and post-translational modifications [15], [16]. However, accurate alignment of separate gels represent a technological challenge for analysis of stacks of 2-D gels and immunoblots [27], [28], in where addition of complex or single protein standards as well as multiple antibodies has provided useful solutions [29], [30], [31], [32], [33].

We have developed two fluorescently labelled protein standards, based on a complex cellular extract and defined non-human proteins, respectively. Both standards improve alignment of 2DI, and the cell based standard enabled the investigation of subtle protein biosignature variation of p53 in leukocytes collected from healthy individuals.