Accurate Multiplexed Proteomics
at the MS2 Level Using
the Complement Reporter Ion Cluster
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Abstract
Isobaric labeling strategies, such as isobaric tags for
relative
and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have
promised to dramatically increase the power of quantitative proteomics.
However, when applied to complex mixtures, both the accuracy and precision
are undermined by interfering peptide ions that coisolate and cofragment
with the target peptide. Additional gas-phase isolation steps, such
as proton-transfer ion–ion reactions (PTR) or higher-order
MS3 scans, can almost completely eliminate this problem. Unfortunately,
these methods come at the expense of decreased acquisition speed and
sensitivity. Here we present a method that allows accurate quantification
of TMT-labeled peptides at the MS2 level without additional ion purification.
Quantification is based on the fragment ion cluster that carries most
of the TMT mass balance. In contrast to the use of low <i>m</i>/<i>z</i> reporter ions, the localization of these complement
TMT (TMT<sup>C</sup>) ions in the spectrum is precursor-specific;
coeluting peptides do not generally affect the measurement of the
TMT<sup>C</sup> ion cluster of interest. Unlike the PTR or MS3 strategies,
this method can be implemented on a wide range of high-resolution
mass spectrometers like the quadrupole Orbitrap instruments (QExactive).
A current limitation of the method is that the efficiency of TMT<sup>C</sup> ion formation is affected by both peptide sequence and peptide
ion charge state; we discuss potential routes to overcome this problem.
Finally, we show that the complement reporter ion approach allows
parallelization of multiplexed quantification and therefore holds
the potential to multiply the number of distinct peptides that can
be quantified in a given time frame