TMTpro™ complementary ions

Under certain conditions, TMTpro labeling can generate strong complementary ions. These are the remnants of the labeled peptides after incomplete MS/MS fragmentation, resulting in the loss of the reporter ion and carbon monoxide. The complementary ion consists of the peptide and the balance region of the label. Using these for quantification is possible, and a suitable workflow was developed by Johnson, A,. et al, TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level, Journal of Proteome Research 20(6):3043–3052 (2021). Complementary ion quantification is limited to 8 channels.

Mascot does not currently support quantification using complementary ions. If they are prominent in your data, their presence may interfere with peptide scoring, as Mascot treats them as unexplained peaks.

TMT_complementary_ions.pl can be used for preprocessing the peak lists before submitting to database searching. The script converts any TMTpro complementary ions into equivalent reporter ions in the standard reporter ion region. The instructions below show how to automate the preprocessing using Mascot Distiller and Mascot Daemon, but the script can also be used on its own.

Mascot Daemon prerequisites

The Mascot Daemon computer will need a copy of Perl installed. The best option at the time of writing is Strawberry Perl. If Mascot Daemon is installed on the same computer as Mascot Server 2.6 or later, this second copy of Perl will not conflict with the version Mascot Server uses. Download and install any recent version of Strawberry Perl, and make sure perl is in the system path. If you are using a Mascot Server version 2.5 or earlier, then the computer already has Perl installed and you don’t need to install another copy.

Download TMT_complementary_ions.zip, extract and save TMT_complementary_ions.pl to a directory on the Mascot Daemon computer.

Data acquisition and peak picking

We normally recommend acquiring TMT data with the instrument in profile mode, so that the MS/MS spectra contain an area measurement under the reporter ions peaks. However, with TMTpro complementary peaks, you need to acquire the MS/MS spectra in centroided mode.

If using Mascot Distiller, please use processing options that treat the MS/MS data as centroided. With Thermo instruments, the default.ThermoXcalibur.opt method is a good set to start with. Treating the data as centroided ensures Distiller does not treat the complementary ion region as peptidic peaks and attempt to deisotope them.

A third-party peak picking program like msconvert can also be used, as it treats the MS/MS spectra as centroided and does not perform any peak picking or deisotoping. You can also use MGF files directly from other peak picking applications generated outside of Mascot Daemon as long as the data has not been deisotoped.

To process the data, set up a Mascot Daemon task with your preferred data import filter or use existing MGF files. Set up an external task to run the script after the MGF file has been created. The command line to use might look like this:

perl "C:\scripts\TMT_complementary_ions.pl" -r -p -d -f "<cachedpeaklist>"

In this example, the TMT_complementary_ions.pl script has been saved in C:\scripts\. It will remove singly charged and doubly charged (-d) TMTpro complementary ions (-p) and convert them into reporter ions (-r). It is very important to check the “Wait for competition” box so that the MGF file is completely processed before Daemon moves on to the search.

Search parameters and quantitation method

The TMTpro quantitation method shipped with Mascot is not suitable for complementary ion quantitation. It is originally configured to identify reporter ions separated by the difference in 13C and 15N isotopes. Go to your local Mascot Server home page, Configuration Editor, Quantitation. Make a copy of the TMTpro method. In the Protocol tab, the Reporter Tolerance and Reporter Tolerance Unit values default to 20ppm. Clear the fields to make the tolerance unspecified. Mascot Server will now use the MS/MS tolerance and unit with the converted reporter ion peaks. You should also edit the protein ratios accordingly; please refer to the reporter ion masses table further below.

Viewing the results

Quantitation results should be viewed on the Mascot Server in the Protein Family Summary report. Results can be exported to other formats in the usual way. Mascot Distiller supports viewing TMTpro results and other reporter ion protocols directly. However, when using TMT_complementary_ions.pl as described above, the peak lists have been modified after Distiller processing. When Distiller imports the search results, it tries to match them to the original peak lists. The transformed reporter ions not present in the original spectra, so no quantitation in Distiller is possible.

TMT_complementary_ions.pl usage help

The command line options for TMT_complementary_ions.pl are as follows:

-f filename

Input data filename. When Mascot Daemon is using Distiller as the data import filter, use "<cachedpeaklist>" as the file name. If you are using pre-existing MGF files, use "<datafilepath>\<datafilename>".

-r

By default, the script detects and removes complementary ions. If -r is specified, the script also converts the removed peaks into reporter ions.

-p

If given, process TMTpro complementary ions. Otherwise, TMT complementary ions.

-d

If given, use the doubly charged complementary ions if present. Otherwise, only singly charged ions are used.

-l

Enable logging. The logging mode records a log to the same directory as the MGF file. The scripts records all the precursor m/z, MH+ and charge along with the calculated complementary ion windows and the number of peaks in each window. It also saves a copy of the original peak list. If logging is switched off, the original peak list is removed. The log is saved as a .tsv file (tab-separated values), which can be opened in any spreadsheet program.

When -r is specified, the script converts complementary ions into reporter ions as specified in the table below.

Mass	Labels
133	126, 127C
132	127N, 128N, 128C
131	129N, 129C
130	130N, 130C
129	131N
128	131C
127	132N, 132C, 133C
126	133N, 134N

As TMTpro complementary ion quantification is limited to 8 channels, the overlap in mass between certain labels does not matter.