The fractions are then analyzed by liquid chromatography tandem mass spectrometry, with the resultant mass spectra providing sequence information, and relative quantification. In an effort to identify novel proteins that are associated with the metastatic progression of human Albaspidin-AA prostate cancer, we have performed a 4-plex iTRAQ analysis using two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 which have different metastatic potentials. PC3-N2 is lowly metastatic while PC3-ML2 is highly metastatic. Following comparative quantitative data analysis, a number of candidates were found to be significantly differentially expressed in PC3-ML2 cells compared to PC3-N2 cells. Two of the candidates identified as being significantly upregulated in PC3-ML2 are plectin and vimentin. These proteins were further investigated by Western blot and siRNA knockdown. The expression levels correlated to immunohistochemistry images from normal and adenocarcinoma prostate tissue samples. The differentially regulated proteins identified in the study, including plectin and vimentin, are discussed in the context of their significance to prostate cancer progression. Cell migration is crucial for the invasion of cancer cells. Plectin interlinks intermediate filaments with microtubules and microfilaments and anchors intermediate filaments to desmosomes or hemidesmosomes. It could be involved in tumor cell migration and invasion which has been shown in several cancers. Vimentin is required to maintain the architecture of the cytoplasm in cells. Vimentin intermediate filaments, in addition to their potential interactions with microfilaments and microtubules, participate in many other specialized cell functions. During tumor metastasis, cancer cells undergo epithelial�Cmesenchymal transition in which epithelial tumor cells in the primary cancer site are converted into aggressive and metastatic tumor cells. One of the main characteristics of EMT is the combination of loss of cell�Ccell contact, reduced E-cadherin expression, and enhanced expression of mesenchymal markers such as vimentin. The mesenchymal-like cells 3,4,5-Trimethoxyphenylacetic acid generated are transported to metastatic sites, where they may undergo mesenchymal�Cepithelial transition by regaining E-cadherin expression, allowing cell�Ccell adhesion and connecting adjacent cells to form new foci. To determine whether plectin and vimentin are involved invasion and migration and thus potentially prostate cancer metastasis, we transfected PC3-ML2 with siRNA targeting the two genes. The transfected cells were used in scratch/wound healing assays to determine the consequences of the gene knockdowns. In these experiments the extent of wound closure is a direct measure of cell motility or migration capacity. In the cells transfected with the control non-targeting siRNA the wound closure was almost complete within 24 hours of transfection in the PC3-ML2 cells. In contrast the wound was still present in PC3-ML2 cells into which plectin and vimentin siRNA had been introduced. Therefore cell migration was significantly inhibited by both plectin and vimentin knockdown cells compared to the non-targeted siRNA controls. We further examined the effect of plectin and vimentin knockdown on PC3-ML2 cell invasion using the transwell migration assay. Control cells and plectin and vimentin knockdown cells passing through the insert membrane was significantly reduced in plectin and vimentin knockdown invasive cells as compared with the control non-knockdown cells. Percent invasion of plectin and vimentin knockdown cells was significantly lower than that of control siRNA-treated cells. We therefore conclude that a reduction in the levels of both plectin and vimentin produced by the cells resulted in suppressed cell invasion in the prostate cancer cell line.