MSFragger is a fast and comprehensive peptide identification tool for shotgun proteomics that enables over 100-fold speed improvements over existing tools. It uses a novel fragment-ion indexing method to perform open database searches, allowing for the identification of peptides and their modified forms. MSFragger is effective in uncovering differences in modification rates across experimental samples and conditions, and has been shown to increase the number of identified spectra in affinity purification experiments by up to 300%. It also improves false discovery rate (FDR) estimation in proteomics by accounting for unaccounted peptide modifications. MSFragger is platform-independent and can be easily integrated into existing data analysis pipelines. It enables comprehensive profiling of chemical and biological modifications across large-scale proteomics datasets, and has been applied to various proteomics applications, including protein-RNA crosslinking studies and large-scale protein interaction studies. MSFragger's ultrafast performance allows for efficient open searching, which is crucial for accurate peptide identification and FDR estimation. The tool has been shown to identify a large number of peptides with modified forms, and to uncover previously undetected modifications. MSFragger's open search strategy has been demonstrated to be effective in identifying peptides that were previously missed in narrow window searches, and to improve the accuracy of protein inference. The tool has also been shown to be useful in the analysis of modified peptides and in the identification of rare and low abundance modifications. Overall, MSFragger provides a powerful and efficient solution for peptide identification in shotgun proteomics, with the ability to handle large datasets and to improve the accuracy of FDR estimation.MSFragger is a fast and comprehensive peptide identification tool for shotgun proteomics that enables over 100-fold speed improvements over existing tools. It uses a novel fragment-ion indexing method to perform open database searches, allowing for the identification of peptides and their modified forms. MSFragger is effective in uncovering differences in modification rates across experimental samples and conditions, and has been shown to increase the number of identified spectra in affinity purification experiments by up to 300%. It also improves false discovery rate (FDR) estimation in proteomics by accounting for unaccounted peptide modifications. MSFragger is platform-independent and can be easily integrated into existing data analysis pipelines. It enables comprehensive profiling of chemical and biological modifications across large-scale proteomics datasets, and has been applied to various proteomics applications, including protein-RNA crosslinking studies and large-scale protein interaction studies. MSFragger's ultrafast performance allows for efficient open searching, which is crucial for accurate peptide identification and FDR estimation. The tool has been shown to identify a large number of peptides with modified forms, and to uncover previously undetected modifications. MSFragger's open search strategy has been demonstrated to be effective in identifying peptides that were previously missed in narrow window searches, and to improve the accuracy of protein inference. The tool has also been shown to be useful in the analysis of modified peptides and in the identification of rare and low abundance modifications. Overall, MSFragger provides a powerful and efficient solution for peptide identification in shotgun proteomics, with the ability to handle large datasets and to improve the accuracy of FDR estimation.