MSGF+ results without ion intensity of TMT 11 plex data

[sunqiangzai]

Dear dev team,

I am Qiang Sun, an assistant professor at Zhejiang University. I am appreciated to your nice software. I have a quick request about TMT-11plex option because it shows very cost-effective in terms of experimental cost. I use the following commands:

msgf_plus -s NA069TQB101_4.mzML -d human_protein_0526.fasta -t 20ppm
-ti -1,2 -e 1 -protocol 4 -m 0 -inst 1 -addFeatures 1
-minLength 6 -maxLength 50 -minCharge 2 -maxCharge 5
-tda 1 -n 1 -ignoreMetCleavage 0 -mod MSGFPlus_Mods1.txt -ntt 1

Modifications (default: none)

229.1629,*,fix,N-term,TMT6plex
229.1629,K,fix,any,TMT6plex
C2H3N1O1,C,fix,any,Carbamidomethyl

Variable Modifications (default: none)

O1,M,opt,any,Oxidation # Oxidation M
C2H2O,*,opt,Prot-N-term,Acetyl # Acetylation Protein N-term

However, the ion intensity was not calculated for the file even I add the parameter -addFeatures 1. I wonder whether the parameter I used is correct or not.

Thanks for your efforts.

Sincerely,

Qiang Sun

[alchemistmatt]

MS-GF+ interprets MS/MS fragmentation patterns to identify peptides. It does not search for reporter ion intensities. You correctly included the +229.1629 mods so that MS-GF+ can successfully match monoisotopic peptide masses to the mass derived from the observed precursor m/z.

In order to find TMT-11 reporter ion intensities, use MASIC, available at https://github.com/PNNL-Comp-Mass-Spec/MASIC/releases
The parameter file to use is similar to this:
https://github.com/PNNL-Comp-Mass-Spec/MASIC/blob/master/Docs/TMT_Example/TMT10_LTQ-FT_10ppm_ReporterTol0.003Da_2014-08-06.xml
but with

<item key="ReporterIonMassMode" value="16" />

We also have software for merging the MS-GF+ results with the MASIC results; see the MASIC Results Merger at https://github.com/PNNL-Comp-Mass-Spec/MASIC-Results-Merger/releases

Screenshots showing example usage:
https://github.com/PNNL-Comp-Mass-Spec/MASIC-Results-Merger/tree/master/Docs

Although it was originally designed to work with the Peptide Hit Results Processor (PHRP), the Readme says it should work with solely the .tsv file from the MZID to TSV Converter

[sunqiangzai]

MS-GF+ interprets MS/MS fragmentation patterns to identify peptides. It does not search for reporter ion intensities. You correctly included the +229.1629 mods so that MS-GF+ can successfully match monoisotopic peptide masses to the mass derived from the observed precursor m/z.

In order to find TMT-11 reporter ion intensities, use MASIC, available at https://github.com/PNNL-Comp-Mass-Spec/MASIC/releases
The parameter file to use is similar to this:
https://github.com/PNNL-Comp-Mass-Spec/MASIC/blob/master/Docs/TMT_Example/TMT10_LTQ-FT_10ppm_ReporterTol0.003Da_2014-08-06.xml
but with

<item key="ReporterIonMassMode" value="16" />

We also have software for merging the MS-GF+ results with the MASIC results; see the MASIC Results Merger at https://github.com/PNNL-Comp-Mass-Spec/MASIC-Results-Merger/releases

Screenshots showing example usage:
https://github.com/PNNL-Comp-Mass-Spec/MASIC-Results-Merger/tree/master/Docs

Although it was originally designed to work with the Peptide Hit Results Processor (PHRP), the Readme says it should work with solely the .tsv file from the MZID to TSV Converter

The TMT11-plex, why use the value =16 but value=11?

[FarmGeek4Life]

You give the value as '16' because that is what corresponds to TMT 11-plex in the list in the program: https://github.com/PNNL-Comp-Mass-Spec/MASIC/blob/master/clsReporterIons.cs#L15

[sunqiangzai]

Thank you very much, my questions have been solved. I also have another question about my TMT data, since I have several fractions of mzML data from one single TMT-11 plex. Do you suggest I run the mgsf+ for the individual file or combine all the files and run ?

[alchemistmatt]

You give the value as '16' because that is what corresponds to TMT 11-plex in the list in the program: https://github.com/PNNL-Comp-Mass-Spec/MASIC/blob/master/clsReporterIons.cs#L15

See also the "Reporter Ions" table in the Readme at https://github.com/pnnl-comp-mass-spec/MASIC#:~:text=Reporter%20Ions

[alchemistmatt]

Always run MS-GF+ against one instrument file at a time. If you instrument is a Thermo instrument, run MS-GF+ against each .raw file (after converting to .mzML). For other instruments, convert each instrument file to a .mzML file and search each .mzML file with MS-GF+