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Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent \'antibody-independent\' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.

A lack of motor neurons abolishes both neurotrophic factor secretion and contractile activity in muscle, which impairs mass, contractile properties, and fibre-type characteristics of the muscle. However, the molecular pathways that can be stimulated or repressed in the scenario of spinal cord injury remain unknown. We investigated for the first time the transcriptional profile of a young male patient 8 months after spinal cord injury. Adaptive metabolic changes of complete denervated skeletal muscle were revealed. In particular, the main molecular pathways involved include metabolic and proteolitic pathways, mitochondrial and synaptic function, calcium homeostasis, sarcomere and anchorage structures. Our data depict the molecular signalling still present in complete denervated skeletal muscle fibres a few months after spinal cord injury. These data could be of interest also to design a specific therapeutic approach aimed at the electrical-stimulation of severe atrophied skeletal muscle.

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