Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identifica- With well-developed algorithms and computational tools for mass spectrometry (MS) 1 data analysis, peptide-based bottom-up proteomics has gained considerable popularity in the field of systems biology (1-9). Nevertheless, the bottom-up approach is suboptimal for the analysis of protein posttranslational modifications (PTMs) and sequence variants as a result of protein digestion (10). Alternatively, the protein-based top-down proteomics approach analyzes intact proteins, which provides a "bird's eye" view of all proteoforms (11), including those arising from sequence variations, alternative splicing, and diverse PTMs, making it a disruptive technology for the comprehensive analysis of proteoforms (12-24). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for processing data from bottom-up proteomics experiments, the data analysis tools in topdown proteomics remain underdeveloped.The initial step in the analysis of top-down proteomics data is deconvolution of high-resolution mass and tandem mass spectra. Thorough high-resolution analysis of spectra by horn (THRASH), which was the first algorithm developed for the deconvolution of high-resolution mass spectra (25), is still widely used. THRASH automatically detects and evaluates individual isotopomer envelopes by comparing the experimental isotopomer envelope with a theoretical envelope and reporting those that score higher than a user-defined threshold. Another commonly used algorithm, MS-Deconv, utilizes a From the ‡Department of Cell and Regenerative Biology, §Molec-ular Pharmacology Training Program, ¶Human Proteomics Program,

Summary Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly, and, thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously un-reported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction, and suggest therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput mass spectrometry-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization.