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Title: Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer

Abstract

Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI–FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments ofmore » a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. Furthermore, a 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.« less

Authors:
 [1];  [2];  [2];  [2];  [2];  [3];  [2];  [2];  [2];  [1];  [1]; ORCiD logo [4]
  1. Univ. of Wisconsin-Madison, Madison, WI (United States)
  2. Thermo Fisher Scientific, San Jose, CA (United States)
  3. Thermo Fisher Scientific, Cambridge, MA (United States)
  4. Univ. of Wisconsin-Madison, Madison, WI (United States); Morgridge Institute for Research, Madison, WI (United States)
Publication Date:
Research Org.:
Univ. of Wisconsin-Madison, Madison, WI (United States); Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1464629
Alternate Identifier(s):
OSTI ID: 1506654
Grant/Contract Number:  
SC0018409
Resource Type:
Accepted Manuscript
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 90; Journal Issue: 15; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Hebert, Alexander S., Prasad, Satendra, Belford, Michael W., Bailey, Derek J., McAlister, Graeme C., Abbatiello, Susan E., Huguet, Romain, Wouters, Eloy R., Dunyach, Jean -Jacques, Brademan, Dain R., Westphall, Michael S., and Coon, Joshua J. Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.8b02233.
Hebert, Alexander S., Prasad, Satendra, Belford, Michael W., Bailey, Derek J., McAlister, Graeme C., Abbatiello, Susan E., Huguet, Romain, Wouters, Eloy R., Dunyach, Jean -Jacques, Brademan, Dain R., Westphall, Michael S., & Coon, Joshua J. Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer. United States. doi:10.1021/acs.analchem.8b02233.
Hebert, Alexander S., Prasad, Satendra, Belford, Michael W., Bailey, Derek J., McAlister, Graeme C., Abbatiello, Susan E., Huguet, Romain, Wouters, Eloy R., Dunyach, Jean -Jacques, Brademan, Dain R., Westphall, Michael S., and Coon, Joshua J. Tue . "Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer". United States. doi:10.1021/acs.analchem.8b02233. https://www.osti.gov/servlets/purl/1464629.
@article{osti_1464629,
title = {Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer},
author = {Hebert, Alexander S. and Prasad, Satendra and Belford, Michael W. and Bailey, Derek J. and McAlister, Graeme C. and Abbatiello, Susan E. and Huguet, Romain and Wouters, Eloy R. and Dunyach, Jean -Jacques and Brademan, Dain R. and Westphall, Michael S. and Coon, Joshua J.},
abstractNote = {Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI–FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. Furthermore, a 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.},
doi = {10.1021/acs.analchem.8b02233},
journal = {Analytical Chemistry},
number = 15,
volume = 90,
place = {United States},
year = {2018},
month = {7}
}

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Figures / Tables:

Figure 1 Figure 1: Characterization of FAIMS CV settings. All results are from 60 min analyses of K562-derived tryptic peptides, except where noted. (+)FAIMS and (−)FAIMS data were collected from analyses with and without the FAIMS device attached, respectively. Distributions of (a) precursor charge states, (b) unique peptide identifications, and (c) proteinmore » groups across CV settings from −10 to −120 V. (d) Effect of CV on tryptic peptide identifications from multiple organisms. (e) Effect of CV on K562 human cell peptide identifications generated from listed proteases. (f) Effect of reducing the inner electrode (I.E.) temperature on the CV distribution of peptide identifications; the outer electrode was held constant at 100 °C.« less

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