Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 µM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 µM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.-Méplan, C., Johnson, I. T., Polley, A. C. J., Cockell, S., Bradburn, D. M., Commane, D. M., Arasaradnam, R. P., Mulholland, F., Zupanic, A., Mathers, J. C., Hesketh, J. Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

Proteomic analysis reveals field-wide changes in protein expression in the morphologically normal mucosa of patients with colorectal neoplasia.

Models for the pathogenesis of colorectal cancer tend to focus on the localized lesion, with less attention paid to changes in normal-appearing mucosa. Here we used two-dimensional gel electrophoresis and mass spectrometry to define patterns of protein expression in morphologically normal colonic mucosa from 13 healthy subjects, 9 patients with adenomatous polyps, and 9 with cancer. Tumor samples were also compared with the normal mucosa. Systematic gel comparisons identified a total of 839 spots that differed significantly between one or more groups (P < 0.05). Principle component analysis indicated that the first three components accounted for approximately 37% of the total variation and provided clear evidence that flat mucosa from healthy subjects differed significantly from that of patients with polyps or cancer. Sixty-one proteins differed significantly between mucosa from healthy subjects and all other tissue types, and 206 differed significantly between healthy mucosa and polyp mucosa. Several of the proteins showing significant underexpression in tumor tissue were cytokeratins and other cytoskeletal components. In contrast, cytokeratins, including several isoforms of cytokeratin 8, were overexpressed in apparently normal mucosa from polyp and cancer patients compared with mucosa from healthy subjects. These findings indicate that protein expression in the apparently normal colonic mucosal field is modified in individuals with neoplastic lesions at sites distant from the lesion. Recognition and further characterization of this field effect at the molecular level may provide protein biomarkers of susceptibility to colorectal cancer and facilitate development of hypotheses for the role of diet and other environmental factors in its causation.