As at MS1-based quantitation, where we can hyperplex SILAC with mTRAQ for instance, is it possible for similar hyperlexing at MS2-level quantitation? Maybe a 6-plex TMT could be hyperplexed with 4-plex iTRAQ?
Dear Bharath, the difficulty is that iTRAQ and TMT use the same chemistry and thus target the same functional groups (N-termini, e-Lysine). Therefore it is difficult to envision a protocol for sequential labeling that will allow for extended multiplexing (or "hyperplexing"). The SILAC/mTRAQ combination is different in that it is a) introduced at different levels of the workflow per se (metabolic vs. chemical labeling, pre vs. post digest) and targets fully orthogonal structural features. The mose promising road to hyperplexing by MS right now seems to be the neutron encoded hyperplexing coming out of the Gygi group (see PubMed 22457332), however this requires VERY high resolution MS which currently is not available to most people.
Thanks Christof! I thought the same.
Yes, I know about NeuCode @ MS1 level (theoretically, it is possible to construct 39 Lys isotopologues, they say) from Coon's group (pmid: 23435260) . Also, as you say they need very high mass resolution of about more than 200,000. So it is possible only if you have an orbitrap elite with developer's kit. Seems to be impossible for non-core proteomics researchers in the near future.
So the only possibility at present seems to be to do SILAC at MS1 level and TMT/iTRAQ at MS2 level, requiring a MS3 scan.
Dear Bharath,
there are different types of TMT reagents available e.g. iodoTMT 6plex.
For quantitation on MS1 level you could use the isotopic version of TMT as used normally for targeted SRM studies (Byers et al, J Proteomics. 2009).
For quantitation on MS2 level you then select the cysteine-reactive iodoTMT 6plex (isobaric) (Qu et al, J Proteome Res. 2014). Therefore you omit targetting the same functional group with TMT (isotopic label at N-termini and Lysine, isobaric at Cysteine)!
Using this design would allow you at least a twelve plex assay.
Maybe you should take a look at the review of Rauniyar & Yates, J Proteome Res. 2014 (PMID: 25337643) where they nicely summarized the different types of isobaric labelling tags and its recommended approaches.
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