Absolute Quantification of Toxicological Biomarkers via Mass Spectrometry.

With the advent of "-omics" technologies there has been an explosion of data generation in the field of toxicology, as well as many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Traditionally, these validation experiments have been conducted using antibody-based technologies such as Western blotting, ELISA, and immunohistochemistry. However, this often produces a significant bottleneck as the time, cost, and development of successful antibodies are often far outpaced by the generation of targets of interest. In response to this, there recently have been several developments in the use of triple quadrupole (QQQ) mass spectrometry (MS) as a platform to provide quantification of proteins. This technology does not require antibodies; it is typically less expensive and quicker to develop assays and has the opportunity for more accessible multiplexing. The speed of these experiments combined with their flexibility and ability to multiplex assays makes the technique a valuable strategy to validate biomarker discovery.

Differential proteomics incorporating iTRAQ labeling and multi-dimensional separations.

Considerable effort is currently being expended to integrate newly developed "omics"-based approaches (proteomics, transcriptomics, and metabonomics) into preclinical safety evaluation workflows in the hope that more sensitive prediction of toxicology can be achieved as reported by Waters and Fostel (Nat. Rev. Genet. 5(12):936-948, 2004) and Craig et al. (J. Proteome Res. 5(7):1586-1601, 2006). Proteomic approaches are well placed to contribute to this effort as (a) proteins are the metabolically active products of genes and, as such, may provide more sensitive and direct predictive information on drug-induced liabilities and (b) they have the potential to determine tissue leakage markers in peripheral fluids. Here, we describe a workflow for proteomic semi-quantitative expression profiling of liver from rats treated with a known hepatotoxicant using a multiplexed isobaric labeling strategy and multi-dimensional liquid chromatography.

Absolute quantification of toxicological biomarkers by multiple reaction monitoring.

With the advent of "-omics" technologies, there has been an explosion of data generation in the field of toxicology, as well as in many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Traditionally, these validation experiments have been conducted using antibody-based technologies such as Western blotting, ELISA, and immunohistochemistry. However, this often produces a significant bottleneck as the time, cost, and development of successful antibodies are often far outpaced by the generation of targets of interest. In response to this, recently there have been several developments in the use of triple quadrupole (QQQ) mass spectrometry (MS) as a platform to provide quantification of proteins by multiple reaction monitoring (MRM). This technology does not require antibodies; it is typically less expensive and quicker to develop, and has the opportunity for more accessible multiplexing. The speed of these experiments combined with their flexibility and ability to multiplex assays makes the technique a valuable strategy to validate biomarker discovery.

Prioritization of candidate protein biomarkers from an in vitro model system of breast tumor progression toward clinical verification.

The use of in vitro cell culture model systems has revealed many potential mediators and candidate biomarkers of various disease phenotypes. To be of clinical utility, the expression of these candidates must be assessed in patient samples such as tissue, urine or blood. However, typical "omic" experiments may produce candidates in such large numbers that it is usually impossible to test all of these in clinical samples. Here, we present a proteomic approach to discover and prioritize candidate biomarkers that are more likely to be found in serum. Using a combination of experimental and in silico approaches, we have demonstrated this approach using an isogenic cell culture model of breast cancer invasion. Differential proteomics (2D-DIGE) was used to discover a number of candidate biomarkers and a subset of these were identified as "extracellular". We tested the validity of this approach by screening serum from breast cancer patients for these candidates and then verified the presence of several of these "extracellular" proteins. This approach provides a pragmatic approach to prioritizing candidates that may be most suitable for downstream assays such as multiple reaction monitoring.

Identification and functional validation of therapeutic targets for malignant melanoma.

Despite remarkable effort, malignant melanoma still remains a potent killer. Millions of dollars have been spent on clinical trials that have not succeeded in achieving significant patient benefit. The thorough validation of drug targets at an earlier stage is, therefore, an essential step in the development of new therapies. Since the development of microarray experiments, putative drug targets are being identified in a high-throughput manner. Though high-throughput functional validation methods are currently being established, a more specific, pre-clinical analysis of promising target genes remains inevitable. For this, a broad range of increasingly sophisticated functional models is available. In vitro, the microenvironment of skin can be simulated through various two or three-dimensional models. In vivo assays range from xenograft studies to the establishment of transgenic organisms. Here, we provide a summary of functional interrogation approaches in melanoma research, focusing on the application of these strategies to the development of new effective therapies.

Breast cancer proteomics: clinical perspectives.

Breast cancer is the one of leading causes of cancer-related deaths in women within economically developed regions of the world. A major focus of present research into this malignancy is the identification of new biomarkers and drug targets to improve detection and treatment. Proteomics represents one of the latest technological developments in this context. It aims to analyse the complex circuitry of the breast cancer proteome. Here, the authors review how breast cancer proteomics has progressed so far, with emphasis on its potential application to clinically relevant scenarios.