Quantitation: Reporter protocol

This is quantitation based on the relative intensities of fragment peaks at fixed m/z values within an MS/MS spectrum. All of the required information contained within the peak list, so the quantitation report can be generated as part of a standard Mascot result report.

Chemistries

iTRAQ™

The first commercially available chemistry for the reporter protocol was Applied Biosystems (now AB SCIEX) iTRAQ. The original reagents provided a set of 4 isobaric tags, and an 8-plex chemistry is now available. A good overview of 4-plex iTRAQ is provided by Ross, P. L., et al., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents, Molecular & Cellular Proteomics 3 1154-1169 (2004). The iTRAQ pages on the AB Sciex web site contain additional information.

TMT® and TMTpro™

The reporter protocol also supports Tandem Mass Tags, as described in Thompson, A., et al., Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS, Analytical Chemistry 75 1895-1904 (2003). TMT is available in 6-plex and 10/11-plex chemistries. The related TMTpro 16-plex is described in Thompson, A., et al., TMTpro: Design, Synthesis, and Initial Evaluation of a Proline-Based Isobaric 16-Plex Tandem Mass Tag Reagent Set, Analytical Chemistry 91(24):15941–15950 (2019), and later expanded to 18-plex.

Configuration notes

The mass tolerance to be used for matching reporter ion peaks can be specified using Reporter Tolerance (reporter_tol, optional) and Reporter Tolerance Unit (reporter_tol_unit, optional). If not specified, the MS/MS tolerance and unit are used.

Precursor ion selection on any TOF instrument tends to have limited resolution, which increases the chance of getting a mixed MS/MS spectrum due to interference from nearby precursor. When this happens, it is likely to make the measured ratio closer to 1. Although the quality element includes a test for whether the precursor region is clean (Fraction threshold), this cannot be used here because the reporter protocol works off the MS/MS peak list, so there is no mechanism for inspecting the survey scan. On the other hand, having a wide precursor selection window can be an advantage, because a narrow window might discriminate against under-enriched precursors. The isotope correction factors are designed to compensate for any under-enrichment, but this assumes that the transmission window is wide enough to include the under-enriched precursors, which are not isobaric with the fully enriched precursor.

iTRAQ modification of Tyr should be included as a variable modification and an exclusion element used to remove these matches from the quantitation report. This is because the reaction with Tyr is slow, and likely to be incomplete, which could lead to inaccurate ratios if these matches were included.

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 2.8 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.

AB SCIEX 4000 / 5000 Series Explorer

When you submit a Mascot search from AB SCIEX 4000 / 5000 Series Explorer or GPS Explorer, the peak areas are not the same as those used by GPS Explorer for quantitation. The same is true you export a peak list using the Peaks to Mascot function. This could be an issue if you plan to perform iTRAQ quantitation outside of GPS Explorer.

TS2Mascot is a simple, free utility to export peak lists with accurate reporter ion peak areas from an AB SCIEX 4000 / 5000 Series database. The peak list can be saved as a Mascot Generic Format (MGF) file or a Mascot search form can be invoked and the peak list submitted directly to Mascot.

TS2Mascot will run under Microsoft Windows NT 4.0 SP6, Windows 2000, Windows XP Professional, and Windows 2003 Server (32 bit only). TS2Mascot communicates with the AB SCIEX 4000 / 5000 Series database using ODBC. The PC will need to have Oracle client software installed, including the Oracle ODBC driver. To submit a Mascot search from TS2Mascot, Microsoft Internet Explorer 4.01 SP2 or later must be installed and operational.

TS2Mascot

To install TS2Mascot, run the Windows installer package. Unless 4000 Series Explorer or GPS Explorer is already installed on the target PC, you may need to create a local Oracle service name, as described in the setup instructions.

Example

Click here for an iTRAQ example of the reporter protocol. We are grateful to Phillip Humphryes and Robert Graham of the Institute of Cancer Sciences at the University of Manchester for permission to use this data, which was acquired on an ABSciex 5600.

You may notice occasional extreme or negative ratios at the peptide level. This is usually where a reporter ion peak has been missed completely in a weak spectrum. The application of the isotope purity correction then "corrects" the intensity to a negative value. It would be easy to hide such ratios, but we feel it is better to display them because a large number of instances might indicate that the peak detection settings require attention. Negative ratios, along with zero and infinity ratios, are discarded when calculating a protein ratio.

To see the quantitation method details, follow the method details link in the report header. Notice how all the modifications have been embedded into the method and an exclusion element used to remove matches with iTRAQ modification on Tyr.