Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye DIGE Cy 3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1 H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.

A novel statistically integrated proteometabonomic method has been developed and applied to a human tumor xenograft mouse model of prostate cancer. Parallel 2D-DIGE proteomic and 1H NMR metabolic profile data were collected on blood plasma from mice implanted with a prostate cancer (PC-3) xenograft and from matched control animals. To interpret the xenograft-induced differences in plasma profiles, multivariate statistical algorithms including orthogonal projection to latent structure (OPLS) were applied to generate models characterizing the disease profile. Two approaches to integrating metabonomic data matrices are presented based on OPLS algorithms to provide a framework for generating models relating to the specific and common sources of variation in the metabolite concentrations and protein abundances that can be directly related to the disease model. Multiple correlations between metabolites and proteins were found, including associations between serotransferrin precursor and both tyrosine and 3-D-hydroxybutyrate. Additionally, a correlation between decreased concentration of tyrosine and increased presence of gelsolin was also observed. This approach can provide enhanced recovery of combination candidate biomarkers across multi-omic platforms, thus, enhancing understanding of in vivo model systems studied by multiple omic technologies.

Purpose:To discover and validate serum glycoprotein biomarkers in ovarian cancer using proteomic-based approaches. Experimental Design: Serum samples from a ''discovery set''of 20 patients with ovarian cancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling.Validation of a candidate biomarker was carried out with Western blotting and immunoassay (n = 424). Results: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing. One of these, confirmed byWestern blotting, was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer. Validation using cross-sectional samples from 303 individuals (healthy controls and patients with benign, borderline, or malignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121 samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases in whom CA-125 was noninformative. Conclusions: Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.