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Shotgun proteomics using liquid chromatography coupled to mass spectrometry (LC-MS) is commonly used to identify peptides containing post-translational modifications. With the emergence of fast database search tools such as MSFragger, the approach of enlarging precursor mass tolerances during the search (termed “open search”) has been increasingly used for comprehensive characterization of post-translational and chemical modifications of protein samples. However, not all mass shifts detected using the open search strategy represent true modifications, as artifacts exist from sources such as unaccounted missed cleavages or peptide co-fragmentation (chimeric MS/MS spectra). Here, we present Crystal-C, a computational tool that detects and removes such artifacts from open search results. Our analysis using Crystal-C shows that, in a typical shotgun proteomics data set, the number of such observations is relatively small. Nevertheless, removing these artifacts helps to simplify the interpretation of the mass shift histograms, which in turn should improve the ability of open search-based tools to detect potentially interesting mass shifts for follow-up investigation.

General Workflow

Figure. Workflow of Crystal-C as applied to each PSM from open search results. (A) Find potential missed cleavage sites by searching the previous and next fully enzymatic peptides of the identified peptide, where MTol is the mass tolerance (20 ppm by default), ME is the precursor neutral mass, MT is the identified peptide mass, and MP and MN are the previous and next adjacent fully enzymatic peptide masses, respectively. (B) Check whether the PSM is semi-enzymatic by deleting one amino acid from the left or right side of the identified peptide sequence at a time and calculating the mass difference between ME and the remaining peptide sequence. If the mass difference is smaller than MTol, the remaining peptide sequence is regarded as semi-enzymatic. (C) Find chimeric MS/MS spectra. Crystal-C searches for peaks from the identified peptide within the isolation window by comparing theoretical isotopic clusters (purple) to the MS1 spectrum. If a peak matching one of the theoretical isotope clusters is found in the isolation window and does not belong to the precursor, the PSM is considered chimeric.


Parameter Description
thread Number of threads. "-1" means that Crystal-C automatically uses (total number of threads - 1) in your computer for processing.
fasta Protein Fasta File
raw_file_location The dictionary where the raw data locates
raw_file_extension The file extension of raw data
output_location The folder for the newly generated pepXML files
precursor_charge The precursor charge state range
isotope_number Number of theoretical isotope peaks need to be generated
precursor_mass Precursor mass tolerance (unit: ppm)
precursor_isolation_window Precursor Isolation Window (unit: Da.)
correct_isotope_error Correct isotope error or not

How to Download

Download the latest version here

How to Cite

Chang HY, Kong AT, da Veiga Leprevost F, Avtonomov DM, Haynes SE, Nesvizhskii AI. Crystal-C: A Computational Tool for Refinement of Open Search Results. J Proteome Res. 2020. Manuscript

For other tools developed by the Nesvizhskii lab, see our website:


(for mzML files) java -Xmx53G -cp "CrystalC-1.2.1.jar" crystalc.Run crystalc.params *.pepXML

(for Thermo RAW files) java"D:\MSFragger-3.0\ext\thermo" -Xmx53G -cp "CrystalC-1.2.1.jar" crystalc.Run crystalc.params *.pepXML


Crystal-C: A computational tool for refinement of open search results




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