TMT Isobaric Labeling: Reporter-Ion Quantification

Sample Channels (true relative abundance)

MS Acquisition

Co-isolation interference 10%

MS2 ion sampling depth (AGC) 1.0×10⁵

Precursor charge state

MS1: all six channels are isobaric — one precursor peak. Click Fragment to release reporter ions.

Readouts

Precursor m/z

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Mean quant. error

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Ch.	true	meas.	err

How it works

A TMT (tandem mass tag) has a reporter group and a balance group joined so their combined mass is constant across the whole plex:

m(reporter_i) + m(balance_i) = 229.163 Da  (constant ∀i)
M_precursor,i = M_peptide + 229.163
m/z = (M_precursor + z·1.00728) / z

Because M_precursor is identical for every channel, chemically identical peptides from up to six (or more) samples co-elute and get isolated together as one MS1 peak — that's the "isobaric" trick that makes multiplexing possible.

During HCD/CID fragmentation the tag's amide bond breaks preferentially, releasing a low-mass reporter ion (nominal 126–131 Da) whose intensity is proportional to how much of that peptide came from its sample. Two real complications are modeled here:

Shot noise — the instrument fills to a target total ion count (AGC). Each channel gets a Poisson-distributed share, so relative precision improves roughly as 1/√N with sampling depth.
Ratio compression — any other peptide accidentally caught in the isolation window contributes an equal background to every reporter channel, pulling measured fold-changes back toward 1:1 even though the true abundances differ (a well-known limitation of isobaric MS2 quantification, mitigated in practice with narrower isolation windows or MS3/SPS).

Move the abundance sliders, add interference, then Fragment to see the reporter-ion bar chart and how far the measured ratios drift from the truth.

MS1 — precursor selection

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