Additional Precursor Purification in Isobaric Mass Tagging Experiments by Traveling Wave Ion Mobility Separation (TWIMS)

Despite
the increasing popularity of data-independent acquisition
workflows, data-dependent acquisition (DDA) is still the prevalent
method of LC–MS-based proteomics. DDA is the basis of isobaric
mass tagging technique, a powerful MS2 quantification strategy that
allows coanalysis of up to 10 proteomics samples. A well-documented
limitation of DDA, however, is precursor coselection, whereby a target
peptide is coisolated with other ions for fragmentation. Here, we
investigated if additional peptide purification by traveling wave
ion mobility separation (TWIMS) can reduce precursor contamination
using a mixture of Saccharomyces cerevisiae and HeLa
proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments,
we find that TWIMS provides a remarkable improvement (on average 2.85
times) in the signal-to-noise ratio for sequence ions. We also report
that TWIMS reduces reporter ions contamination by around one-third
(to 14–15% contamination) and even further (to 6–9%)
when combined with a narrowed quadrupole isolation window. We discuss
challenges associated with applying TWIMS purification to isobaric
mass tagging experiments, including correlation between ion m/z and drift time, which means that coselected
peptides are expected to have similar mobility. We also demonstrate
that labeling results in peptides having more uniform m/z and drift time distributions than observed for
unlabeled peptides. Data are available via ProteomeXchange with identifier
PXD001047.