Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca2+) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.

Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics.

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present challenges to study using conventional structural biology techniques. Here we develop a native nanoproteomics strategy for the enrichment and subsequent native top-down mass spectrometry (nTDMS) analysis of endogenous cardiac troponin (cTn) complex directly from human heart tissue. The cTn complex is enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complex, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the cTn complex, localizes Ca2+ binding domains, defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a paradigm for structural characterization of endogenous native protein complexes.

Structure and dynamics of endogenous protein complexes in human heart tissue captured by native nanoproteomics.

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present tremendous challenges to study using conventional structural biology techniques. Here we develop a "native nanoproteomics" strategy for the native enrichment and subsequent native top-down mass spectrometry (nTDMS) of low-abundance protein complexes. Specifically, we demonstrate the first comprehensive characterization of the structure and dynamics of cardiac troponin (cTn) complexes directly from human heart tissue. The endogenous cTn complex is effectively enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complexes, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the heterotrimeric cTn complex, localizes Ca2+ binding domains (II-IV), defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a new paradigm for structural characterization of low-abundance native protein complexes.

Structure and dynamics of endogenous protein complexes in human heart tissue captured by native nanoproteomics.

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present tremendous challenges to study using conventional structural biology techniques. Here we develop a "native nanoproteomics" strategy for the native enrichment and subsequent native top-down mass spectrometry (nTDMS) of low-abundance protein complexes. Specifically, we demonstrate the first comprehensive characterization of the structure and dynamics of cardiac troponin (cTn) complexes directly from human heart tissue. The endogenous cTn complex is effectively enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complexes, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the heterotrimeric cTn complex, localizes Ca2+ binding domains (II-IV), defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a new paradigm for structural characterization of low-abundance native protein complexes.

Distinct core glycan and O-glycoform utilization of SARS-CoV-2 Omicron variant Spike protein RBD revealed by top-down mass spectrometry.

The SARS-CoV-2 Omicron (B.1.1.529) variant possesses numerous spike (S) mutations particularly in the S receptor-binding domain (S-RBD) that significantly improve transmissibility and evasion of neutralizing antibodies. But exactly how the mutations in the Omicron variant enhance viral escape from immunological protection remains to be understood. The S-RBD remains the principal target for neutralizing antibodies and therapeutics, thus new structural insights into the Omicron S-RBD and characterization of the post-translational glycosylation changes can inform rational design of vaccines and therapeutics. Here we report the molecular variations and O-glycoform changes of the Omicron S-RBD variant as compared to wild-type (WA1/2020) and Delta (B.1.617.2) variants using high-resolution top-down mass spectrometry (MS). A novel O-glycosite (Thr376) unique to the Omicron variant is identified. Moreover, we have directly quantified the Core 1 and Core 2 O-glycan structures and characterized the O-glycoform structural heterogeneity of the three variants. Our findings reveal high resolution detail of Omicron O-glycoforms and their utilization to provide direct molecular evidence of proteoform alterations in the Omicron variant which could shed light on how this variant escapes immunological protection.

Rbm20 ablation is associated with changes in the expression of titin-interacting and metabolic proteins.

Dilated cardiomyopathy (DCM) is a major risk factor for developing heart failure and is often associated with an increased risk for life-threatening arrhythmia. Although numerous causal genes for DCM have been identified, RNA binding motif protein 20 (Rbm20) remains one of the few splicing factors that, when mutated or genetically ablated, leads to the development of DCM. In this study we sought to identify changes in the cardiac proteome in Rbm20 knockout (KO) rat hearts using global quantitative proteomics to gain insight into the molecular mechanisms precipitating the development of DCM in these rats. Our analysis identified changes in titin-interacting proteins involved in mechanical stretch-based signaling, as well as mitochondrial enzymes, which suggests that activation of pathological hypertrophy and altered mitochondrial metabolism and/or dysfunction, among other changes, contribute to the development of DCM in Rbm20 KO rats. Collectively, our findings provide the first report on changes in the cardiac proteome associated with genetic ablation of Rbm20.

Distinct Core Glycan and O-Glycoform Utilization of SARS-CoV-2 Omicron Variant Spike Protein RBD Revealed by Top-Down Mass Spectrometry.

The SARS-CoV-2 Omicron (B.1.1.529) variant possesses numerous spike (S) mutations particularly in the S receptor-binding domain (S-RBD) that significantly improve transmissibility and evasion of neutralizing antibodies. But exactly how the mutations in the Omicron variant enhance viral escape from immunological protection remains to be understood. The S-RBD remains the principal target for neutralizing antibodies and therapeutics, thus new structural insights into the Omicron S-RBD and characterization of the post-translational glycosylation changes can inform rational design of vaccines and therapeutics. Here we report the molecular variations and O-glycoform changes of the Omicron S-RBD variant as compared to wild-type (WA1/2020) and Delta (B.1.617.2) variants using high-resolution top-down mass spectrometry (MS). A novel O-glycosite (Thr376) unique to the Omicron variant is identified. Moreover, we have directly quantified the Core 1 and Core 2 O-glycan structures and characterized the O-glycoform structural heterogeneity of the three variants. Our findings reveal high resolution detail of Omicron O-glycoforms and their utilization to provide direct molecular evidence of proteoform alterations in the Omicron variant which could shed light on how this variant escapes immunological protection.