Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry

Multiplexed,
isobaric tagging methods are powerful techniques to
increase throughput, precision, and accuracy in quantitative proteomics.
The dynamic range and accuracy of quantitation, however, can be limited
by coisolation of tag-containing peptides that release reporter ions
and conflate quantitative measurements across precursors. Methods
to alleviate these effects often lead to the loss of protein and peptide
identifications through online or offline filtering of interference
containing spectra. To alleviate this effect, high-Field Asymmetric-waveform
Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to
reduce precursor coisolation and improve the accuracy and dynamic
range of multiplex quantitation. Here we tested the use of FAIMS to
improve quantitative accuracy using previously established TMT-based
interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed
that FAIMS robustly improved the quantitative accuracy of both high-resolution
MS2 (HRMS2) and synchronous precursor selection
MS3 (SPS-MS3)-based methods without sacrificing
protein identifications. We further optimized and characterized the
main factors that enable robust use of FAIMS for multiplexed quantitation.
We highlight these factors and provide method recommendations to take
advantage of FAIMS technology to improve isobaric-tag-quantification
moving forward.