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Global Phosphoproteome Analysis Using High-Field Asymmetric Waveform Ion Mobility Spectrometry on a Hybrid Orbitrap Mass Spectrometer

  • Laura K. Muehlbauer
    Laura K. Muehlbauer
    National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
  • Alexander S. Hebert
    Alexander S. Hebert
    National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
  • Michael S. Westphall
    Michael S. Westphall
    National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
  • Evgenia Shishkova
    Evgenia Shishkova
    National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
  • , and 
  • Joshua J. Coon*
    Joshua J. Coon
    National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
    Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
    Morgridge Institute for Research, Madison, Wisconsin 53706, United States
    *Email: [email protected]
Cite this: Anal. Chem. 2020, 92, 24, 15959–15967
Publication Date (Web):December 3, 2020
https://doi.org/10.1021/acs.analchem.0c03415
Copyright © 2020 American Chemical Society

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    Abstract

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    Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15–20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c03415.

    • Supplemental materials and methods; (Figure S1) characterization of phosphopeptides across various CVs; (Figure S2) combining various CVs to determine overall phosphosite depth and phosphopeptide diversity; (Figure S3) effect of total cycle time on phosphosite identifications for single-shot FAIMS analyses; (Figure S4) examining phosphopeptides unique to experiments with or without FAIMS; (Figure S5) sampling redundancy between liquid chromatography and gas-phase fractionation; (Figure S6) phosphosite intensity reproducibility by total cycle time; and (Figure S7) relative standard deviation of class I phosphosite intensities from five replicate injections with and without FAIMS (PDF)

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