Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

Andrea F.G. Gargano; Jared B. Shaw; Mowei Zhou; Christopher S. Wilkins; Thomas L. Fillmore; Ronald J. Moore; Govert W. Somsen; Ljiljana Paša-Tolić

doi:10.1021/acs.jproteome.8b00458

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

Andrea F.G. Gargano^*, Jared B. Shaw, Mowei Zhou, Christopher S. Wilkins, Thomas L. Fillmore, Ronald J. Moore, Govert W. Somsen, Ljiljana Paša-Tolić

^*Corresponding author for this work

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Research output: Contribution to Journal › Article › Academic › peer-review

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 ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow 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 proteoforms starting from limited sample quantities (∼1.5 μg).

Original language	English
Pages (from-to)	3791-3800
Number of pages	10
Journal	Journal of Proteome Research
Volume	17
Issue number	11
Early online date	18 Sept 2018
DOIs	https://doi.org/10.1021/acs.jproteome.8b00458
Publication status	Published - 2 Nov 2018

Funding

This work was financially supported by The Netherlands Organization for Scientific Research by the NWO Veni grant IPA (722.015.009). The authors would like to thank Nikola Tolic and Rui Zhao from the Pacific Northwest National Laboratories as well as Andrei Barcaru and Eva M.O.L. Johansson from the University of Amsterdam for their support and valuable discussions. We gratefully acknowledge Dr. Andrew Alpert (PolyLC inc.) for the support in developing the WCX-HILIC method and Hagen Preik-Steinhoff (VICI-Valco) for the kind gift of the nano HPLC valve. This work was performed at EMSL, a national scientific user facility sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy (DOE). EMSL is located at PNNL, a multidisciplinary national laboratory operated by Battelle for the U.S. DOE.

Funders	Funder number
U.S. Department of Energy
Biological and Environmental Research
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	722.015.009

Keywords

histones
online comprehensive two-dimensional liquid chromatography
post-translational modifications
top-down mass spectrometry
ultraviolet photodissociation

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Gargano, A. F. G., Shaw, J. B., Zhou, M., Wilkins, C. S., Fillmore, T. L., Moore, R. J., Somsen, G. W., & Paša-Tolić, L. (2018). Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS. Journal of Proteome Research, 17(11), 3791-3800. https://doi.org/10.1021/acs.jproteome.8b00458

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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 ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow 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 proteoforms starting from limited sample quantities (∼1.5 μg).",

keywords = "histones, online comprehensive two-dimensional liquid chromatography, post-translational modifications, top-down mass spectrometry, ultraviolet photodissociation",

author = "Gargano, \{Andrea F.G.\} and Shaw, \{Jared B.\} and Mowei Zhou and Wilkins, \{Christopher S.\} and Fillmore, \{Thomas L.\} and Moore, \{Ronald J.\} and Somsen, \{Govert W.\} and Ljiljana Pa{\v s}a-Toli{\'c}",

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doi = "10.1021/acs.jproteome.8b00458",

language = "English",

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Gargano, AFG, Shaw, JB, Zhou, M, Wilkins, CS, Fillmore, TL, Moore, RJ, Somsen, GW & Paša-Tolić, L 2018, 'Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS', Journal of Proteome Research, vol. 17, no. 11, pp. 3791-3800. https://doi.org/10.1021/acs.jproteome.8b00458

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS. / Gargano, Andrea F.G.; Shaw, Jared B.; Zhou, Mowei et al.
In: Journal of Proteome Research, Vol. 17, No. 11, 02.11.2018, p. 3791-3800.

Research output: Contribution to Journal › Article › Academic › peer-review

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T1 - Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

AU - Gargano, Andrea F.G.

AU - Shaw, Jared B.

AU - Zhou, Mowei

AU - Wilkins, Christopher S.

AU - Fillmore, Thomas L.

AU - Moore, Ronald J.

AU - Somsen, Govert W.

AU - Paša-Tolić, Ljiljana

PY - 2018/11/2

Y1 - 2018/11/2

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KW - histones

KW - online comprehensive two-dimensional liquid chromatography

KW - post-translational modifications

KW - top-down mass spectrometry

KW - ultraviolet photodissociation

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JO - Journal of Proteome Research

JF - Journal of Proteome Research

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ER -

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

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Keywords

UN SDGs

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