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Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes. We employed this technique to study the proteins expressed in colorectal tissues along the normal-adenoma-carcinoma sequence using a multidimensional peptide separation approach coupled to tandem mass spectrometry (LC-MS/MS). We created pooled lysates of biopsy specimens from healthy colonic mucosa (2 sets), small adenomas (2 sets), large adenomas (1 set), and surgical specimens of carcinoma (3 sets) and adjacent normal tissue (3 sets), each from 20 individuals. Tryptic lysate digests were resolved into 20 fractions by isoelectric focusing (IEF) and analyzed by LC-MS/MS on an LTQ-Orbitrap. Four replicate analyses were done on each pooled tissue digest. Spectra were searched against the IPI human protein database in forward and reverse directions using the Myrimatch search …