Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry

Citation:

Devin K Schweppe, Satendra Prasad, Michael W Belford, Jose Navarrete-Perea, Derek Bailey, Romain Huguet, Mark P Jedrychowski, Ramin Rad, Graeme McAlister, Susan E Abbatiello, Eloy R Woulters, Vlad Zabrouskov, Jean-Jacques Dunyach, Joao A Paulo, and Steven P Gygi. 2019. “Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry.” Anal Chem.

Abstract:

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 co-isolation 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 co-isolation 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.