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

Description:
Single muscle fibers (SMFs) are multinucleated single cells that are functionally classified as either fast- or slow-twitch. Post-translational modifications (PTMs) and isoforms of myofilament proteins within SMFs have profound effects on fiber functional properties. To better understand biology occurring at the single fiber level, methods to analyze proteins within SMFs are needed. 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. Conventional proteomics methods extract proteins from large pieces of muscle, which represents an ensemble measurement of heterogeneous mixture of fast/slow-twitch SMFs, connective tissue, and blood vessels. Therefore, we developed a highly sensitive top-down proteomics strategy, enabled by a one-pot protein extraction, capillary reverse phase chromatography, and a Newomics microflow-nanoelectrospray ionization source, for the analysis of SMF proteoforms.

The data revealed distinct differences in PTMs and isoform expression in SMFs across three different muscles: Vastus Lateralis, Soleus, and Plantaris. The online LC-MS/MS method effectively characterized highly homologous isoforms such as the fast, slow, and ventricular isoforms of myosin light chain 1. Fiber-to-fiber changes in troponin T proteoforms were shown, which demonstrates the diversity of isoform expression at the single fiber level. Lastly, fiber-specific isoforms of myosin heavy chain (223 kDa) were measured for the first time from SMFs and used to classify each SMF. Performing top-down proteomics at the single fiber level provides a more accurate assessment of fiber-specific proteoforms compared to bulk tissue analysis, providing new molecular insights at the single fiber level.

Speaker: Jake Melby - University of Wisconsin-Madison
I am a 5th Year Analytical Chemistry Ph.D. student at the University of Wisconsin-Madison under the supervision of Professor Ying Ge. My graduate thesis goal is to develop novel technologies to address the current challenges that face the top-down proteomics field and apply these methodologies to investigate a wide range of biological questions. In particular, I have contributed to finding solutions that aid in linking proteoforms with function, as well as addressing the challenge of analytical sensitivity of intact protein MS . The highly interdisciplinary nature of my thesis work ultimately seeks to provide new technologies, knowledge, and methods for the advancement of top-down proteomics in biomedical research.