Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS
Abstract
Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultra-modified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nano-flow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e. charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e. hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteforms starting from limited sample quantities (~1.5 µg).
- Authors:
-
[1];
[2];
[2];
- Center for Analytical Sciences Amsterdam (The Netherlands); Vrije Universiteit Amsterdam (The Netherlands)
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1507723
- Report Number(s):
- PNNL-SA-138847
Journal ID: ISSN 1535-3893
- Grant/Contract Number:
- AC05-76RL01830
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Proteome Research
- Additional Journal Information:
- Journal Volume: 17; Journal Issue: 11; Journal ID: ISSN 1535-3893
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; histones; online comprehensive two-dimensional liquid chromatography; post-translational modifications; top-down mass spectrometry; ultraviolet photodissociation
Citation Formats
Gargano, Andrea F. G., Shaw, Jared B., Zhou, Mowei, Wilkins, Christopher S., Fillmore, Thomas L., Moore, Ronald J., Somsen, Govert W., and Pasa Tolic, Ljiljana. Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS. United States: N. p., 2018.
Web. doi:10.1021/acs.jproteome.8b00458.
Gargano, Andrea F. G., Shaw, Jared B., Zhou, Mowei, Wilkins, Christopher S., Fillmore, Thomas L., Moore, Ronald J., Somsen, Govert W., & Pasa Tolic, Ljiljana. Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS. United States. doi:10.1021/acs.jproteome.8b00458.
Gargano, Andrea F. G., Shaw, Jared B., Zhou, Mowei, Wilkins, Christopher S., Fillmore, Thomas L., Moore, Ronald J., Somsen, Govert W., and Pasa Tolic, Ljiljana. Tue .
"Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS". United States. doi:10.1021/acs.jproteome.8b00458. https://www.osti.gov/servlets/purl/1507723.
@article{osti_1507723,
title = {Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS},
author = {Gargano, Andrea F. G. and Shaw, Jared B. and Zhou, Mowei and Wilkins, Christopher S. and Fillmore, Thomas L. and Moore, Ronald J. and Somsen, Govert W. and Pasa Tolic, Ljiljana},
abstractNote = {Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultra-modified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nano-flow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e. charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e. hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteforms starting from limited sample quantities (~1.5 µg).},
doi = {10.1021/acs.jproteome.8b00458},
journal = {Journal of Proteome Research},
number = 11,
volume = 17,
place = {United States},
year = {2018},
month = {9}
}
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