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Proteoform Identification pp 15–30Cite as

Size Exclusion Chromatography Strategies and MASH Explorer for Large Proteoform Characterization

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2500))

Abstract

Top-down mass spectrometry (MS)-based analysis of larger proteoforms (>50 kDa) is typically challenging due to an exponential decay in the signal-to-noise ratio with increasing protein molecular weight (MW) and coelution with low-MW proteoforms. Size exclusion chromatography (SEC) fractionates proteins based on their size, separating larger proteoforms from those of smaller size in the proteome. In this protocol, we initially describe the use of SEC to fractionate high-MW proteoforms from low-MW proteoforms. Subsequently, the SEC fractions containing the proteoforms of interest are subjected to reverse-phase liquid chromatography (RPLC) coupled online with high-resolution MS. Finally, proteoforms are characterized using MASH Explorer, a user-friendly software environment for in-depth proteoform characterization.

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Acknowledgments

We would like to thank Trisha Tucholski for helpful discussions. This work was supported by NIH R01 GM117058 and NIH R01 GM125085 (to YG). YG also would like to acknowledge R01 HL096971 and S10 OD018475. TNT would like to acknowledge support from the NIH Chemistry-Biology Interface Training Program, T32 GM008505. JAM would like to acknowledge support from the Training Program in Translational Cardiovascular Science, T32 HL007936-20.

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Authors and Affiliations

  1. Department of Chemistry, University of Wisconsin – Madison, Madison, WI, USA

    Timothy N. Tiambeng, Zhijie Wu, Jake A. Melby & Ying Ge

  2. Department of Cell and Regenerative Biology, University of Wisconsin – Madison, Madison, WI, USA

    Ying Ge

  3. Human Proteomics Program, University of Wisconsin – Madison, Madison, WI, USA

    Ying Ge

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  1. Timothy N. Tiambeng
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  2. Zhijie Wu
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  3. Jake A. Melby
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  4. Ying Ge
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Correspondence to Ying Ge .

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Editors and Affiliations

  1. Department of Chemistry, Michigan State University, East Lansing, MI, USA

    Liangliang Sun

  2. Deming Department of Medicine, School of Medicine, Tulane University, New Orleans, LA, USA

    Xiaowen Liu

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Tiambeng, T.N., Wu, Z., Melby, J.A., Ge, Y. (2022). Size Exclusion Chromatography Strategies and MASH Explorer for Large Proteoform Characterization. In: Sun, L., Liu, X. (eds) Proteoform Identification. Methods in Molecular Biology, vol 2500. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2325-1_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2325-1_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2324-4

  • Online ISBN: 978-1-0716-2325-1

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