Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry.
Devin K SchweppeSatendra PrasadMichael W BelfordJose Navarrete-PereaDerek J BaileyRomain HuguetMark P JedrychowskiRamin RadGraeme McAlisterSusan E AbbatielloEloy R WoultersVlad ZabrouskovJean-Jacques DunyachJoão A PauloSteven P GygiPublished in: Analytical chemistry (2019)
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.
Keyphrases
- mass spectrometry
- high resolution
- liquid chromatography
- high performance liquid chromatography
- gas chromatography
- ms ms
- capillary electrophoresis
- high resolution mass spectrometry
- tandem mass spectrometry
- multiple sclerosis
- single cell
- endothelial cells
- social media
- liquid chromatography tandem mass spectrometry
- binding protein
- crispr cas
- single molecule
- induced pluripotent stem cells
- molecular dynamics
- small molecule