Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry
Open accessAbstract
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 H uman- Y east P rot e omics R esource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS 2 (HRMS 2 ) and synchronous precursor selection MS 3 (SPS-MS 3 )-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.
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- Authors
- Schweppe, D. K., Prasad, S., Belford, M. W., Navarrete-Perea, J., Bailey, D. J., Huguet, R., Jedrychowski, M. P., Rad, R., McAlister, G., Abbatiello, S. E., Woulters, E. R., Zabrouskov, V., Dunyach, J.-J., Paulo, J. A., & Gygi, S. P.
- Affiliations
- Harvard University (US)
- Thermo Scientific LSMS, San Jose, California 95134, United States
- Corresponding author
- Schweppe, Devin K.
- Gygi, Steven P.
- ORCID
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-01-23
- Volume
- 91
- Issue
- 6
- Pages
- 4010-4016
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC10777686
- OpenAlex
- W2912956724
- Cited by
- 229
- References
- 20
- Citations by year
- 2026: 25 · 2025: 34 · 2024: 36 · 2023: 40 · 2022: 24 · 2021: 32 · 2020: 32 · 2019: 6
- FWCI
- 9.36
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Human Genome Research Institute (U24 HG006673 07)
- National Institute of Diabetes and Digestive and Kidney Diseases (K01 DK098285)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- FAIMS, isobaric tagging, Trail Making Test, dynamic range, quantitative accuracy, method optimization
- MeSH terms
- Humans, Neoplasm Proteins, Peptides, Saccharomyces cerevisiae, Mass Spectrometry, Proteome, Saccharomyces cerevisiae Proteins, Proteomics, HCT116 Cells
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