Quantitation: Reporter protocol
The Reporter protocol 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 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). See also the Applied Biosystems iTRAQ™ Reagents document on the Thermo Fisher website.
TMT® and TMTpro™
The reporter protocol also supports Tandem Mass Tags, as described by 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). The most recent addition is TMTpro 18-plex, Li, J., et al., TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing, Journal of Proteome Research 20(5):2964-2972 (2021).
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), the threshold 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.
Example: TMTpro 18-plex
The raw data used in the TMTpro-18plex publication is available as PRIDE project PXD024275. For simplicity and to keep the example file size small, we selected a single raw file, ea05139_02042021_phos_T18.raw, which is the phosphopeptide enriched, unfractionated sample.
The TMTpro 18-plex quantitation method shipped with Mascot works fine, but the default ratios are not meaningful in this experiment. We copied TMTpro 18-plex and renamed to “TMTpro 18plex PXD024275″. In this method, the first nine channels are normalised to “DMSO-”, which is the average of channels 129C, 130N and 130C. These three channels are biological replicates of DMSO in PTEN-/- cell lines. The last nine channels are similarly normalised to “DMSO+”, which is the average of channels 134N, 134C and 135N. These are the biological replicates of DMSO in PTEN+/+.
The raw file was processed with Mascot Distiller. The MS1 scans are profile and MS2 scans centroid data, so default.ThermoXcalibur.opt is a suitable starting point. The default reporter ion window must be widened to accommodate TMTpro 18-plex. In Distiller, go to Processing, Edit processing options and MS/MS Peak Picking tab. Uncheck “Same as MS peak picking”, which is the default with default.ThermoXcalibur.opt, and check “Pick single peaks in this range”. Increase the upper mass limit from 130.5 to 135.5.
After processing all scans, the data were searched against the human proteins in SwissProt and a contaminants database. Precursor and fragment tolerances suggested in the paper were 50ppm and 0.9Da, but an initial Mascot search suggests both MS1 and MS2 are high accuracy. Precursor tolerance 10ppm and fragment tolerance 0.02Da gave the best results. We also set #13C to 1, as the vendor peak detection has sometimes chosen the 13C peak rather than 12C.
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 how the quantitation ratios are defined.
