Quantitative Analysis Of Subcellular Proteins In Colorectal Cancer

Part One:Differential quantitative cytoskeleton protein expression of colorectal cancer using an iTRAQ labeling-based proteomics approachBackground:Subcellular organelles and structures include cellular membrane, mitochondrion, Golgi, nucleus, et al. It is known that organelles are undergoing dynamic remodeling. Cytoskeleton is a dynamic network consisting of various protein polymers and regulatory proteins. Cytoskeleton proteins is of great importance to cellular shape, cellular mobility, inter-cellular and extra-cellular signaling.Purpose:By quantitatively comparing cytoskeleton protein expression of colorectal cancer (CRC)and matched normal mucosa using an iTRAQ labeling-based proteomics approach, we tried to explore and discover colorectal-specific differentially expressed cytoskeleton proteins.Methods:Primary CRC tissues and matched normal adjacent normal colorectal tissues were obtained from20patients undergoing surgical resection from November2010to December2010at affiliated tumour hospital of Fudan University, China. Cytoskeleton proteins were extracted, purified, quantified, enzymolysis and pooled. iTRAQ labeling coupling with2DLC-MS/MS approach was employed to differentiate and quantify labeled peptides. GO analysis and protein network analysis were performed using bioinformatics tools.Results:A total of67differentially expressed cytoskeleton proteins were indentified and quantified. Among them,10proteins were significantly up-regulated and15were significantly down-regulated. These proteins were of vital importance in biological process including cell adhesion and migration, cell growth, dissemination and their regulations.Part two:Subcellular localization of ANXA3and PKM2of colorectal cancerBackground:It is hoped that combining with more advanced technologies these MS-based approaches will uncover many novel, specific CRC marker candidates. Nonetheless, one of the biggest challenges in this area is to identify the subcellular localization of those differentially expressed proteins and to understand whether these dynamic changes in protein localizations can improve early detection of CRC.Purpose:The goal of this investigation was to determine the subcellular localization of PKM2and ANXA3in stage I and II CRC by profiling their protein expression patterns in human CRC tissues and matched normal colonic tissues and correlating these with patient clinical-pathological data.Methods:Primary colorectal cancer tissues and matched normal adjacent normal colorectal tissues were obtained from20patients undergoing surgical resection from November2010to December2010at affiliated tumour hospital of Fudan University, China. Cytosolic/membranes and membrane organelles/nuclear enriched proteins were sequentially extracted and quantified. Differential expression of PKM2and ANXA3were detected using Western blot.Results:1PKM2was significantly up-regulated in cytosol of Stage I CRC compared to adjacent normal mucosa.ANXA3as significantly up-regulated in membrane/organelle of Stage I CRC compared to adjacent normal mucosa.2PKM2was predominantly located in cytosol in stage I CRC, while it was predominantly located in membrane/organelle in stage II CRC.3We found no significant relation between subcellular location of PKM2and clinical-pathological data.