To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-beta-induced EMT. Further analysis of cofilin1 and beta-actin revealed an increase in their mRNA stability in response to TGF-beta, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-beta-induced EMT.

ProteomeCommons.org has implemented a resource that incorporates concepts of Web 2.0 social networking for collaborative annotation of data sets placed in the Tranche repository. The annotation tools are part of a project management resource that is effective for individual laboratories or large distributed groups. The creation of the resource was motivated by the need for a way to encourage annotation of data sets with high accuracy and compliance rates. The system is designed to respond to the dynamic nature of research in an easy-to-use fashion through the use of a dynamic data model that does not inhibit the innovation that is important for basic research. Placing the annotation tool within a project manager allows annotation to occur over the life of the project and provides the security and monitoring capabilities needed for large or small collaborative projects. The resource effectively supports distributed groups of investigators working on common data sets and is available immediately at https://ProteomeCommons.org. In addition, a silver compliant data resource based on ProteomeCommons.org has been developed for cancer Biomedical Informatics Grid (caBIG) to allow much broader access to the annotations describing data sets in the Tranche repository.

The fragmentation characteristics of peptides derivatized at the side-chain -amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C -N bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n Ϫ 1) ϩ -charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzylmodified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary -amino group of the benzyl-derivatized lysyl side chain. If the secondary -amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary -amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways. Specifically, the inability to predict the relative intensities of sequence product ions produced reduces the confidence of peptide sequence assignments when data from MS/MS spectra are searched against immense lists of in silico peptide sequences generated from proteome databases [2][3][4]. This represents a barrier to the development of more effective computational tools for accurately assigning peptide sequences to MS/MS spectra as most database search tools are practically limited to treating the formation of all possible product ions with equal probabilities. An improved understanding of the detailed mechanisms involved in peptide fragmentation with CID will directly lead to more effective search tools. Not surprisingly, peptide fragmentation by CID has been studied extensively, and these efforts have culminated in a number of theoretical models that explain many of the observed fragmentation properties of peptides [1]. One example, the "Pathways in Competition" model [1], is associated with the mobilization of ionizing protons [5] to the amide backbone, competitive cleavages of the amide bonds, and post-cleavage proton transfer and chemical reactions. This model currently represents the most comprehensive description of the mechanism leading to the formatio...