Quantitation: Multiplex protocol

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 precursor components are not isobaric, and the peaks used for quantitation are the sequence ions that include the labelled terminus. To be suitable for 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. That is, if the delta was 6 Da, (e.g. SILAC), 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".
• 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.
• The peptide ratio is calculated from the sums of all the valid multiplex ion intensities for each component. This means that each peptide ratio is a weighted average of the multiplex peaks.

Configuration notes

• 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)
• Exclude internal label should be checked if there is the possibility that a peptide could have a label on a residue that is not at the terminus. For example, if the labelling is K and R followed by digestion with trypsin, a internal label would occur if there was a missed cleavage. (exclude_internal_label, optional, default true)
• 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.

Modifications

The code for the multiplex protocol has been written in a very general way, and supports more than 2 components and multiple modifications per component. However, there have to be a number of rules to keep the problem 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. Modification specificity must be consistent with multiplex terminus
3. The components must have complementary modifications
4. Cannot have same modification in multiple components
5. Either the specificity is a list of residues anywhere, or a list of residues at terminus, or a terminus; it cannot be a mix of these specificities
6. Specificity (position) cannot be Protein N-term or Protein C-term
7. Within a component, each residue and terminus can only appear once

Example

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,