Proteogenomic analysis reveals unanticipated adaptations of colorectal tumor cells to deficiencies in DNA mismatch repair.

A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We used standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data.

Integrative omics analysis reveals the importance and scope of translational repression in microRNA-mediated regulation.

MicroRNAs (miRNAs) are key post-transcriptional regulators that inhibit gene expression by promoting mRNA decay and/or suppressing translation. However, the relative contributions of these two mechanisms to gene repression remain controversial. Early studies favor a translational repression-centric scenario, whereas recent large-scale studies suggest a dominant role of mRNA decay in miRNA regulation.

Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior.

GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc.

A Reporter System for Translational Readthrough of Stop Codons in Human Cells.

Agents to induce readthrough of premature termination codons (PTCs) are useful research tools and potential therapeutics. Reporters used to detect PTC readthrough are gene-specific and thus are not suited to for general assessment of readthrough activity or in cases where PTC-inactivated genes are unknown. Here we describe a GFP-based reporter construct pMHG-W57* which is capable of detecting dose-dependent drug-induced PTC readthrough both by fluorescence microscopy and flow cytometry.

Protein identification using customized protein sequence databases derived from RNA-Seq data.

The standard shotgun proteomics data analysis strategy relies on searching MS/MS spectra against a context-independent protein sequence database derived from the complete genome sequence of an organism. Because transcriptome sequence analysis (RNA-Seq) promises an unbiased and comprehensive picture of the transcriptome, we reason that a sample-specific protein database derived from RNA-Seq data can better approximate the real protein pool in the sample and thus improve protein identification. In this study, we have developed a two-step strategy for building sample-specific protein databases from RNA-Seq data.

Proteomic consequences of a single gene mutation in a colorectal cancer model.

The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors.

IDPicker 2.0: Improved protein assembly with high discrimination peptide identification filtering.

Tandem mass spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared among multiple proteins.

Reactive aldehyde modification of thioredoxin-1 activates early steps of inflammation and cell adhesion.

Several lines of evidence suggest that an increase in aldehyde-modified proteins is associated with development of atherosclerosis. Acrolein and 4-hydroxynonenal (HNE) are reactive aldehydes generated during active inflammation as a consequence of lipid peroxidation; both react with protein thiols, including thioredoxin-1 (Trx1), a protein recently found to regulate antioxidant function in endothelial cells. The present study examined whether acrolein or HNE modification of Trx1 could potentiate monocyte adhesion to endothelial cells, an early event of atherosclerosis.

Selective oxidative stress in cell nuclei by nuclear-targeted D-amino acid oxidase.

The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H(2)O(2))-producing enzyme, D-amino acid oxidase (DAAO), to the nucleus (NLS-DAAO) and used this to generate H(2)O(2) in the nuclei of cells. On addition of N-acetyl-D-alanine (NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed.

Compartmental oxidation of thiol-disulphide redox couples during epidermal growth factor signalling.

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling.