High Sensitivity Top-down Proteomics Reveals Heterogeneous Proteoform Profiles Amongst Various Single Skeletal Muscle Fibers

Jake A. Melby¹, David S. Roberts¹, Kyle A. Brown¹, Yutong Jin¹, Eli J. Larson¹, Emily Chapman¹, Yanlong Zhu^{2 3}, Guillaume Tremintin⁴, Daojing Wang⁵, Gary M. Diffee⁶, Ying Ge^{1 2 3}

1. Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
2. Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53705, USA
3. Human Proteomics Program, University of Wisconsin-Madison, Madison, WI 53705, USA
4. Bruker Daltonics, San Jose, CA 94538, USA
5. Newomics Inc., Berkeley, CA 94710, USA
6. Department of Kinesiology, University of Wisconsin-Madison, Madison, WI 53706, USA

Abstract

Single muscle fibers (SMFs) are multinucleated single cells that are classified as fast- and slow-twitch fibers. Post-translational modifications (PTMs) and isoforms of myofilament proteins within SMFs have profound effects on fiber functional properties. To better understand how PTMs and isoforms affect fiber function, it is important to analyze myofilament proteins within SMFs instead of their constituent muscles, which contain a heterogeneous mixture of fast/slow-twitch SMFs, connective tissue, and blood vessels. Top-down mass spectrometry (MS)-based proteomics is a premier technology for the analysis of proteoforms – protein products that arise from single genes due to events such as genetic mutations, alternative splicing, and PTMs. Due to the inherent challenges associated with top-down proteomics, conventional methods extract proteoforms from large pieces of muscle, which convolutes the relationship between proteoforms and SMF function. Herein, we have developed a highly sensitive top-down proteomics strategy, using the Newomics MnESI source, for the analysis of SMF proteoforms revealing distinct fiber-to-fiber proteoform profiles.

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