This study investigates the specificity of trypsin in proteomics experiments, focusing on its ability to cleave peptides exclusively C-terminal to arginine or lysine residues. The authors used a high-accuracy ion trap Fourier transform mass spectrometer (LTQ-FT) to achieve sub-parts per million (ppm) mass accuracy, significantly enhancing the confidence in peptide identification compared to typical ion trap experiments. They found that non-tryptic peptides, which are often reported in published studies, occur only as C-terminal peptides of proteins or as breakdown products of fully tryptic peptides with an N-terminal proline. The study concludes that trypsin indeed exclusively cleaves C-terminal to arginine and lysine residues, and that previous reports of non-tryptic peptides should be re-evaluated. The findings have important implications for improving the reliability of peptide and protein identifications in proteomics research.This study investigates the specificity of trypsin in proteomics experiments, focusing on its ability to cleave peptides exclusively C-terminal to arginine or lysine residues. The authors used a high-accuracy ion trap Fourier transform mass spectrometer (LTQ-FT) to achieve sub-parts per million (ppm) mass accuracy, significantly enhancing the confidence in peptide identification compared to typical ion trap experiments. They found that non-tryptic peptides, which are often reported in published studies, occur only as C-terminal peptides of proteins or as breakdown products of fully tryptic peptides with an N-terminal proline. The study concludes that trypsin indeed exclusively cleaves C-terminal to arginine and lysine residues, and that previous reports of non-tryptic peptides should be re-evaluated. The findings have important implications for improving the reliability of peptide and protein identifications in proteomics research.