Tandem Mass Tags
Isobaric tags for relative and absolute quantitation (iTRAQ) or Tandem Mass Tags (TMT) is a quantitation method in which peptide N-terminus and side chain amines are covalently labeled with tags of varying masses. Up to 16 different samples or treatments can be analyzed using this technique. After labeling and pooling of all the samples, they are analyzed by LC-MS/MS or LC-MS/MS/MS in a single run. The fragmentation of the attached tags produces a low molecular mass reporter ions that are used to relatively quantify the peptides and the proteins from which they originated. The chemical structures of all the tags are identical but each contains isotopes substituted at various positions. Upon fragmentation quantitation data are simultaneously obtained from fragmentation of the tags, giving rise to mass reporter ions. In an LC-MS run peptides labeled with different tags are indistinguishable and the total signal of a peptide observed in MS-mode (MS1) will be the summed signal of peptides labelled with each of the unique tags.
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Problems with co-isolation which can “dampen” signal – fix: MS2-MS3 (ref) but this lower bandwidth.
Use 0ff-line fractionation. – for example basic reversed phase.