HCD/CID spectra merger tool
About
HCD/CID spectra merger tool is a tool to merge the peptide sequence-ion m/z range from CID spectra and the reporter-ion m/z range from HCD spectra into the appropriate single file, to be further used in identification and quantification search engines. Additionally it also separates both spectral data into two different files.
The tool can be used from the ExPASy website) or downloaded for local usage (see below).
The tool can be used from the ExPASy website) or downloaded for local usage (see below).
Reference
Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags.
Loïc Dayon, Carla Pasquarello, Christine Hoogland, Jean-Charles Sanchez, Alexander Scherl.
Journal of Proteomics (2010) 73(4): 769-777.
DOI:10.1016/j.jprot.2009.10.015 PMID: 19903544
DOI:10.1016/j.jprot.2009.10.015 PMID: 19903544
Overview
It has been shown (see reference above) that combining CID and HCD activation modes in a linear ion trap / orbitrap (LTQ-OT) hybrid instrument allows precise peptide quantification without compromising peptide identification with the linear ion trap analyzer. To do so, a higher-energy HCD spectrum and a low energy CID spectrum are acquired on each selected precursor ion. The CID spectrum is then used for peptide identification at highest sensitivity, and the HCD spectrum is used for precise peptide quantification. HCD/CID spectra merger tool is a tool to create composite spectra containing both qualitative and quantitative information to be further used in classical MS/MS search engines (such as Phenyx, Mascot, etc.). Because this program can also dissociate CID and HCD spectra, it can be used for different data analysis schemes, as for example a separated analysis pipeline for CID and HCD spectra.
Basically it kept, for a given precursor ion, the CID fragments (m/z and ion abundance), and added to their mass list the HCD fragments (m/z and ion abundance) comprised in a specified m/z window (typically in the zone of reporter-ions). The resulting mgf file can be uploaded in your favorite identification or quantification software.
Basically it kept, for a given precursor ion, the CID fragments (m/z and ion abundance), and added to their mass list the HCD fragments (m/z and ion abundance) comprised in a specified m/z window (typically in the zone of reporter-ions). The resulting mgf file can be uploaded in your favorite identification or quantification software.
Input
- File (mgf): the mgf file containing CID/HCD spectra
- Number of CID scans: the number of precursor ions selected in a data-dependent manner for each scan cycle (for example: 1 MS1 survey scan followed by 3 data-dependent MS2 scans by CID in the ion trap and the same 3 data-dependent MS2 scans by HCD in the orbitrap equals to 3 CID scans). The CID scans have to be acquired before the HCD scans!
- Peptide masses range to be kept for HCD (TMT / iTRAQ): the lower and upper range of peptide masses to be kept in the merged resulting file.
Output
The tool will provide 3 separate files:
- One file including merged HCD/CID spectra named with extension '_merged.mgf'
- One file for HCD spectra only named with extension '_onlyHCD.mgf'
- One file for CID spectra only named with extension '_onlyCID.mgf'
Download
The script may be downloaded free of charge from here. It requires Perl (5.6 or higher), freely available here.
Usage: perl HCD_CID_merger.pl <filename (mgf)> <CID scan nb> <peptide masses lower range> <peptide masses upper range>
The output files are created in the same directory where the original mgf is located.
Usage: perl HCD_CID_merger.pl <filename (mgf)> <CID scan nb> <peptide masses lower range> <peptide masses upper range>
The output files are created in the same directory where the original mgf is located.
Contact
For questions, suggestions or comments,
please contact us.