MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

Multiplexed quantitation via isobaric
chemical tags (e.g., tandem
mass tags (TMT) and isobaric tags for relative and absolute quantitation
(iTRAQ)) has the potential to revolutionize quantitative proteomics.
However, until recently the utility of these tags was questionable
due to reporter ion ratio distortion resulting from fragmentation
of coisolated interfering species. These interfering signals can be
negated through additional gas-phase manipulations (e.g., MS/MS/MS
(MS3) and proton-transfer reactions (PTR)). These methods, however,
have a significant sensitivity penalty. Using isolation waveforms
with multiple frequency notches (i.e., synchronous precursor selection,
SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby
increasing the number of reporter ions in the MS3 spectrum 10-fold
over the standard MS3 method (i.e., MultiNotch MS3). By increasing
the reporter ion signals, this method improves the dynamic range of
reporter ion quantitation, reduces reporter ion signal variance, and
ultimately produces more high-quality quantitative measurements. To
demonstrate utility, we analyzed biological triplicates of eight colon
cancer cell lines using the MultiNotch MS3 method. Across all the
replicates we quantified 8 378 proteins in union and 6 168
proteins in common. Taking into account that each of these quantified
proteins contains eight distinct cell-line measurements, this data
set encompasses 174 704 quantitative ratios each measured in
triplicate across the biological replicates. Herein, we demonstrate
that the MultiNotch MS3 method uniquely combines multiplexing capacity
with quantitative sensitivity and accuracy, drastically increasing
the informational value obtainable from proteomic experiments.