Two-dimensional linear ion trap mass spectrometers are rapidly becoming the new workhorse instruments for shotgun proteomic analysis of complex peptide mixtures. The objective of this study was to compare the potential for false positive peptide sequence matches between a two-dimensional ion trap instrument and a traditional, three-dimensional ion trap instrument. Through the comparative analysis of a complex protein sample, we found that in order to minimize false positive sequence matches, sequence match scoring criteria must be more stringent for data from the two-dimensional ion trap compared to the three-dimensional ion trap data. Given this increased potential for false positives, we also investigated two potential filtering strategies to reduce the false positive matches for data derived from the two-dimensional ion trap, including trypsin enzyme cleavage filtering, and the addition of peptide physicochemical information as a constraint, specifically peptide isoelectric point. The results described here provide a cautionary tale to researchers, demonstrating the need for careful analysis of MS/MS data from this new class of ion trap instruments, as well as the effectiveness of trypsin enzyme cleavage filtering and peptide pI information in maximizing high confidence protein identifications from this powerful proteomic instrumentation.