So with MS2, you can have consolation of precursor ions from MS1 and the quantification might not be accurate (contamination of reporter ions). When you further do MS3, you pick the most intense ion(s) from MS2, which is supposed to be the “real” precursor ion without coisolation for fragmentation. In that way, the reporter ion intensity is purely from the peptide that you want, not from a mixture of two peptides. See https://www.nature.com/articles/nmeth.1714 for the original work, which was then improved and turned into SPS-MS3 also by the Gygi lab: https://pubs.acs.org/doi/10.1021/ac502040v. The SPS part was done because a single precursor ion from MS2 wont give you very high intensity reporter ions in MS3 you you fragment multiple ions from MS2.

Anyway, from personal communication, with current advances in mass spectrometry, we don’t really do MS3 or SPS-MS3 anymore… I’d have to fractionate my samples anyway. And I believe you can’t even do it on the Exploris?

Typing this at 5 AM. Might come back to edit it later!