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Liquid chromatography paired with tandem mass spectrometry is a standard technique for identifying peptides from complex protein mixtures. Most fragment ion spectra acquired by this technique are unique, but some are repeated. Similarities among the spectra from 1D and 2D liquid chromatography experiments were calculated by the dot product algorithm. Similar spectra were grouped, and the degree of duplication was calculated for each sample. In 1D liquid chromatography data from 1D gel bands, 18% of the fragment ion spectra were duplicates. A six-cycle 2D liquid chromatographic separation of more than 200 proteins produced 28% duplicate spectra. A rat hippocampal homogenate analyzed by a 12-cycle 2D liquid chromatographic separation contained 25% duplicate spectra. Removal of these duplicate spectra, however, resulted in fewer peptides being successfully identified by SEQUEST. We propose a modification for peptide identification algorithms that would improve their performance and accuracy by explicitly recognizing and making use of spectral similarity.