MS PepSearch

This program was developed for proteomics and metabolomics applications, in which hundreds or thousands of unknown tandem mass spectra are generated. The program works by searching a peak list file against up to 4 MS/MS reference libraries. MSPepSearch may input centroided mass spectra in MGF and MSP formats. The output is a match list in tab-delimited text format which can easily be viewed in a spreadsheet application or parsed with scripts as part of a pipeline. To speed-up the searches and properly weight the scoring algorithm, many modifications to the program have been made.

MSPepSearch is intended for evaluation and use by researchers interested in identification of peptides (i.e., mainly those generated by digestion with trypsin) and metabolites by mass spectral library searching. You can download the NIST Peptide MS/MS libraries on this page.

The peptide software does not contain the NIST/EPA/NIH Mass Spectral Library for interpretation of EI mass spectra. However, MSPepSearch.exe console application is fully compaible with NIST EI and small molecules MS/MS libraries.

Download and Installation Instruction

MSPepSearch is a simple Windows program that allows to select data files for processing, select parameters and run the search against spectral libraries.

  • 1. Download the MSPepSearch uppropriate for your Windows operation system. If you are in doubt, select 32-bit version.
  • 2. Extract the MSPepSearch zip archive in a working directory on your hard drive (e.g., C:\projects\MSPepSearch).
  • 3. Download one or more peptide libraries in 'NIST' format from here and extract them to a directory of your choice.

Operating Instructions

(click on the picture to see bigger image)

The main window of the program can be divided into 4 parts:

  • 1. List of input data: The program currently reads input spectra in MGF and MSP file formats. User can select either individual files or an entire folder that contains MGF and/or MSP files.
  • 2. Output directory selection: User should select output directory (folder) where the program will save results. By default the output directory is set to the folder for the first selected data file/folder. Output (report) file is a tab delimited text file. The extension of the output file is “.tsv”. The name of each report file is identical to the name of the input file from which it was generated with the “tsv” extension appended to it. For example report file for input file named ‘test.mgf’ will be named ‘test.mgf.tsv’.

When all input files are processed the program will automatically open the directory in which the output files are located. The output files are best viewed in a spread sheet program.

  • 3. List of libraries to use: User can select up to 4 peptide libraries to be used
  • 4. Options: User can select multiple options that will affect how the search is conducting. The description is below.


  • Pre-search Mode: Fast pre-search mode is default. The standard mode is slower and intended for use with large libraries.
  • m/z Limits: Compare the peaks within the ranges specified by the minimum and maximum values (Min.≤ m/z ≥ Max.). Setting maximum value to -1 sets the maximum value to infinity
  • Search Tolerance Settings: Precursor ion tolerance specifies the precursor ion uncertainty in m/z units. The default value is 1.6. Peaks within m/z units of this value will be ignored if Ignore peaks around precursor check box is checked. Fragment peak m/z tolerance specifies fragment ion mass tolerance. The default value is 0.8.
  • Other Settings
    • Minimum match factor (MF) to output: The output report will contain the scores equal or above this value. The default is set to 450 (a reasonable similarity threshold)
    • Minimum normalized peak intensity: It acts as noise filter when searching.
    • Maximum number of output hits: Default value of 3 will limit the output to the top 3 matches per spectrum
    • Show spectra without matches: Will report spectrum identifiers without match.
    • Include precursor m/z in the output: Will add “Precursor m/z” to report file.
    • Include Hit-Unknown precursor m/z difference: Adds an extra column “Delta(m/z)”-precursor mass error to the report file. The Precursor mass error is in m/z units.
    • Output the input spectrum number: Adds an extra column called “Num” to the report file.
    • Calculate rev-dot: Adds an extra column called “Rev-Dot” to the report file.
    • Use number of replicates: Includes number of replicates for a spectrum in calculating hit probability.
    • Q-TOF: To use the scoring corrections appropriate for the Q-TOF data.
    • Set program priority above normal: Elevates program execution priority from “normal” to “above normal” (use with caution).
    • Load libraries in memory: Allows to increase the performance speed of the program.

Restrictions and Disclaimers

MSPepSearch program may be redistributed without restriction.

Peptide mass spectral libraries are freely available. However, the libraries may not be redistributed without permission. The National Institute of Standards and Technology (NIST) uses its best efforts to deliver high-quality reference data and software products. However, NIST makes no warranties to that effect; and NIST shall not be liable for any damage that may result from the use of the above mentioned products.

Certain commercial equipment, instruments, or materials are identified in this document. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the products identified are necessarily the best available for the purpose.

peptidew/mspepsearch.txt · Last modified: 2014/10/21 16:30 (external edit)

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