The role of toxicoproteomics in assessing organ specific toxicity

Abstract

Aims of this chapter on the role of toxicoproteomics in assessing organ-specific toxicity are to define the field of toxicoproteomics, describe its development among global technologies, and show potential uses in experimental toxicological research, preclinical testing and mechanistic biological research. Disciplines within proteomics deployed in preclinical research are described as Tier I analysis, involving global protein mapping and protein profiling for differential expression, and Tier II proteomic analysis, including global methods for description of function, structure, interactions and post-translational modification of proteins. Proteomic platforms used in toxicoproteomics research are briefly reviewed. Preclinical toxicoproteomic studies with model liver and kidney toxicants are critically assessed for their contributions toward understanding pathophysiology and in biomarker discovery. Toxicoproteomics research conducted in other organs and tissues are briefly discussed as well. The final section suggests several key developments involving new approaches and research focus areas for the field of toxicoproteomics as a new tool for toxicological pathology.

Figure 1

Disciplines of toxicoproteomics to study effects of drug, chemical, disease or environmental stressor exposure. Proteomic analysis attempts to describe various protein attributes in a global manner. Tier I proteomic analysis involves protein mapping or profiling. Protein mapping for identification reflects the property of primary amino acid sequence; quantitations of all proteins from a defined space are inherent in protein profiling; and isolation or enrichment of proteins from a particular spatial location within cells or tissues help to characterize the organism's phenotype. Tier II proteomic analysis involves global determination of individual protein attributes (behavior and structure) regarding their three-dimensional structures, post-translational modifications, functional capabilities, and interactions and complexation with other biomolecules. In protein mapping, the underlined portions of an individual protein represent tryptic peptides for amino acid sequencing for identification by matrix-assisted laser desorption/ionization (MALDI) or tandem mass spectrometry (MS/MS). In protein profiling, changing levels of individual proteins (bar graph) or groups of proteins (cluster analysis) are measured over treatment (T1, T2, T3) or time. A proteome of interest occupies a specific spatial location for analysis and may comprise a subcellular organelle, tissue or organ. Protein structure may represent the β-pleated sheet or α-helix to form tertiary or quaternary protein folding. Specific post-translational moieties, such as ubiquitin (Ubi), phosphorylation (PO₄), glycosylation (GlcNAc), or chemical adduct, are covalently bound to specific amino acid residues on the protein that impart important functional and biophysical properties. Protein function may be: enzymatic, such as enzymatic (E) conversion of substrate (S) to product (P); structural, providing form and shape; translocational, across cells or tissues; signaling and transduction; or many other utilities to be carried out within cells and tissues. Protein interactions may occur between other proteins, between DNA and proteins, or between other biomolecules.

Figure 2

Proteomic platforms for toxicoproteomics studies. Proteomic platforms represent strategies for global separation and identification of proteins. Separations are generally accomplished by gel electrophoresis in toxicoproteomic studies, although more recent studies incorporate liquid chromatography (LC)-based platforms, such as linear column gradients or multidimensional chromatography (MuDPIT). Use of stable isotopes greatly facilitates protein quantitation (ICAT, isotope coded affinity tags; iTRAQ, isobaric tags for relative and absolute quantitation; SILAC, stable isotope labeling by amino acids in cell culture). Label-free methods such as multiple reaction monitoring (MRM) or spectral counting provide protein quantitation without stable isotopes (see text for further explanation). Mass spectrometry (MS) is the primary tool as a means of protein identification in proteomic analysis. Identification occurs by peptide mapping or amino acid (AA) sequencing. Retentate chromatography MS has been used for rapid profiling of biofluid samples using chemically reactive surfaces for separation and MALDI for generating protein mass spectra (i.e., SELDI technology). However, alternatives to MS-based identification in proteomic analysis exist in platforms based upon affinity arrays such as (A) antibody arrays, (B) antibody multiplexing and (C) fluorescently tagged antibody bound to bead suspensions such as the Luminex technology.