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Quantitation: Multiplex protocol

Multiplex is quantitation based on the relative intensities of sequence ion fragment peaks within an MS/MS spectrum. The original approach, described in Zhang, G. A. and Neubert, T. A., Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling, Molecular & Cellular Proteomics 5 401-411 (2006), used a chemistry that labelled one peptide terminus and had a reasonably small mass shift.

Multiplex is very different from the reporter protocol, even though both use MS/MS fragment peaks. In the reporter protocol, there is a single set of reporter ion peaks at fixed m/z. In multiplex, the peaks used for quantitation are the sequence ions that include the labelled terminus. To be suitable for the original, single-terminus multiplex, a chemistry must meet the following requirements:

  • The label must reside at either the C-terminus or N-terminus of the peptides. The label can be residue independent, like 18O labelling of the C-terminus carboxyl group, or a residue label that is constrained to the terminus, such as SILAC modification of K and R, followed by an efficient tryptic digest.
  • The mass shift introduced by the label needs to be small because both labelled and unlabelled precursors must be transmitted through "MS1" simultaneously. Since the selection window could be centered on either the light or heavy precursor, the half width of the transmission window needs to be their m/z separation plus the width of the isotope envelope plus a margin of safety. For example, if the delta was 6 Da for a SILAC label, and singly charged precursors were excluded, the transmission window would still need to be at least 8 m/z. In fact, the wider, the better, because this is likely to make the central transmission region more "flat-topped".
    Note that the mass tolerance for the Mascot search should be the usual one for the instrument. The m/z value for the selected precursor will correspond to either the heavy or the light peptide, and will be matched as such, even though both heavy and light are being transmitted and the MS/MS spectrum is for the mixture.
  • More than two components will be tough unless the spectra are very clean and the fragment ion resolution is high. Otherwise, interference between isobaric fragment ions will reach unacceptable levels. (We have never encountered a data set with more than 2 components, so the code has not been fully tested for this possibility.)
  • The peptide ratio is calculated from the sums of all the valid multiplex ion intensities for each component.

More recently, Isobaric Peptide Termini Labeling (IPTL) has been described in Koehler, C. J., et al., Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics, J. Proteome Research 8 4333-4341 (2009). This labels both termini and the difference between the two components is a mass increase at one terminus exactly balanced by a mass decrease at the other. For example, one component might be Succinyl d0 at the N-terminus and IMID d4 at C-terminal K while the other is Succinyl d4 and IMID d0. Having isobaric precursors removes the requirement for the transmission window between MS1 and MS2 to be wide enough to accommodate the mass shift introduced by the label.


For multiplex, Mascot is being asked to match a mixed MS/MS spectrum, which is usually not a good idea. The matching can be improved by including an artificial neutral loss in each modification definition, equivalent to the complete modification mass. This allows Mascot to match both the labelled and unlabelled fragment ion peaks.

Modifications defined at component level, which identify the ion series to be used for quantitation, must have N-terminus or C-terminus specificity. Sometimes, this will necessitate using a local definition within the quantitation method, even though the modification exists in Unimod, but with a site specificity of Anywhere. For example, the SILAC modification Label:13C(6) appears in the modifications list as Label:13C(6) (K) but not as Label:13C(6) (C-term K). In most cases, you have to create a local definition in any case, because of the unusual neutral loss requirements.

The code for the multiplex protocol has been written in a very general way, but with a number of rules to keep the task manageable. These are mostly enforced in the report script, rather than the search engine or the XML schema:

  1. Exactly one modification group per component
  2. The components must have complementary modifications (i.e. same set of specificities)
  3. Modification specificity must be terminus or residue at terminus
  4. Cannot have same modification in multiple components
  5. Specificity cannot be Protein N-term or Protein C-term
  6. Within a component, each residue and terminus can only appear once

Configuration notes

This implementation of the multiplex protocol contains several of the precautions found in Zhang and Neubert’s original ms2ratio script:

  • It is important to eliminate sequence ions that have a potential overlap from a complementary series. The set of ion series to be considered is defined by the choice of INSTRUMENT. Assuming that the Exclude isobaric fragments option is checked, (and it would be highly inadvisable to clear this), any peaks in the multiplex series that have calculated overlaps from any other series, will be discarded. Clearly, it is very important to choose or define an appropriate INSTRUMENT.
    The testing for isobaric interference uses the same fragment ion tolerance as the Mascot search. In addition, if the 13C peak of a non-multiplex series ion matches a multiplex ion, this is also treated as a match, (but not vice versa). (exclude_isobaric_fragments, optional, default true)
  • Ratios calculated from low intensity peak pairs will usually be less accurate than those from high intensity peaks; partly due to counting statistics and partly due to background. The Ion intensity threshold is used to remove weak peaks. This parameter is the cut-off threshold as a fraction of the intensity of the strongest peak in the multiplex series. For example, if the multiplex series was y, the strongest y ion had an intensity of 1000, and the Ion intensity threshold was 0.1, any pair of y ions where both peaks were below 100 would be discarded. (ion_intensity_threshold, optional, default 0.1)
  • Minimum ion pairs should be set to something sensible. (min_ion_pairs, optional, default 4)
  • If the separation between light and heavy components is small, then it is important to specify an averagine correction to compensate for the overlap between the isotope envelopes. An impurity correction for incomplete labelling may also be required.

Restricting modifications to those with N-terminus or C-terminus specificity was a change introduced in Mascot 2.3, as part of the implementation of IPTL. This has made Exclude internal label redundant, and its setting is ignored (exclude_internal_label, optional, default true).


Click here for an example of the multiplex protocol. To see the quantitation method details, follow the method details link in the report header or click here. We are grateful to Guoan Zhang and Thomas A. Neubert of the Skirball Institute of Biomolecular Medicine for permission to use this data, which forms part of a study of signaling proteins in the EphB2 Pathway. NG108-EphB2 cells were cultured in [13C6]Lys / [13C6]Arg medium or control medium with amino acids of natural isotopic abundance. For full details of the sample work-up, and interpretation of the results, refer to the original publication. Note that this example report is a small extract from the complete dataset.

As expected, contaminant proteins, such as trypsin, human keratin, and BSA, which are unlabelled, show very low ratios, close to zero. One protein, EphB2, is strongly up-regulated. Click on the link for query 13 to display a peptide view report. The pairs for y(5), y(6), and y(7) show the heavy / light ratio very clearly.