Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry.

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates, such as fatty acids, carbohydrates, lipids, and amino acids, to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities, which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection: direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF-MS/MS). Using DI-FTICR MS, 9644 metabolic features were detected where 7156 were assigned a molecular formula and 1107 were annotated by accurate mass assignment. Using LC-Q-TOF-MS/MS, 21,428 metabolic features were detected where 285 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 1340 heart metabolites were identified in this study, which span a wide range of polarities including polar (benzenoids, carbohydrates, and nucleosides) as well as nonpolar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results from this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.

MASH Native: a unified solution for native top-down proteomics data processing.

MOTIVATION: Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. RESULTS: We have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a "one-stop shop" for characterizing both native protein complexes and proteoforms. AVAILABILITY AND IMPLEMENTATION: The MASH Native app, video tutorials, written tutorials, and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry.

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates such as fatty acids, carbohydrates, lipids, and amino acids to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic extractions) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection - direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF MS/MS). Using DI-FTICR MS, 9,521 metabolic features were detected where 7,699 were assigned a chemical formula and 1,756 were assigned an annotated by accurate mass assignment. Using LC-Q-TOF MS/MS, 21,428 metabolic features were detected where 626 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 2276 heart metabolites were identified in this study which span a wide range of polarities including polar (benzenoids, alkaloids and derivatives and nucleosides) as well as non-polar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results of this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.

MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing.

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php . All data files shown in user tutorials are included with the MASH Native software in the download .zip file.