Biology subjects

Melby, J. A.

Publications and source records attributed to Melby, J. A..

7 recordsLinked to original sources

bioRxiv2023

Native nanoproteomics captures the structure and dynamics of endogenous protein complexes in human heart tissue

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 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.

biochemistry↗

bioRxiv2023

Lactate and Immunomagnetic-purified iPSC-derived Cardiomyocytes Generate Comparable Engineered Cardiac Tissue Constructs

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACs-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. After purification, hiPSC-CMs were combined with hiPSC-cardiac fibroblasts to create 3D hiPSC-ECT constructs maintained in culture for four weeks. There were no structural differences observed, and there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force, Ca2+ transients, and {beta}-adrenergic response revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in any protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable molecular and functional properties, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/539642v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@54b370org.highwire.dtl.DTLVardef@d0bbc8org.highwire.dtl.DTLVardef@1d847e3org.highwire.dtl.DTLVardef@578c1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Comprehensive Characterization of Endogenous Phospholamban Proteoforms Enabled by Photocleavable Surfactant and Top-down Proteomics

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modification (PTMs). In particular, membrane proteins play critical roles in cellular functions and represent the largest class of drug targets. However, the top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their intrinsic hydrophobicity and low abundance. Phospholamban (PLN) is a regulatory membrane protein located in the sarcoplasmic reticulum and is essential for regulating cardiac muscle contraction. PLN has diverse combinatorial PTMs and their dynamic regulation has significant influence on cardiac contractility and disease. Herein, we have developed a rapid and robust top-down proteomics method enabled by a photocleavable anionic surfactant, Azo, for the extraction and comprehensive characterization of endogenous PLN from cardiac tissue. We employed a two-pronged top-down MS approach using an online reversed-phase liquid chromatography tandem MS (LC-MS/MS) method on a quadrupole time-of-flight (Q-TOF) MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance (FTICR) MS. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous human cardiac PLN. We have shown the site-specific localization of phosphorylation to Ser16 and Thr17 by MS/MS for the first time and the localization of S-palmitoylation to Cys36. Taken together, we have developed a streamlined top-down targeted proteomics method for comprehensive characterization of combinatorial PTMs in PLN toward better understanding the role of PLN in cardiac contractility.

biochemistry↗

bioRxiv2023

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.

bioinformatics↗

bioRxiv2022

High Sensitivity Top-down Proteomics Captures Single Muscle Cell Heterogeneity in Large Proteoforms

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy to enable the classification of individual fiber types. This study represents the first "single-cell" top-down proteomics analysis that captures single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems. Significance StatementSingle-cell technologies are revolutionizing biology and molecular medicine by allowing direct investigation of the biological variability among individual cells. Top-down proteomics is uniquely capable of dissecting biological heterogeneity at the intact protein level. Herein, we develop a highly sensitive single-cell top-down proteomics method to reveal diverse molecular variations in large proteins (>200 kDa) among individual single muscle cells. Our results both reveal and characterize the differences in protein post-translational modifications and isoform expression possible between individual muscle cells. We further integrate functional properties with proteomics and accurately measure myosin isoforms for individual muscle fiber type classification. Our study highlights the potential of top-down proteomics for understanding how single-cell protein heterogeneity contributes to cellular functions.

biochemistry↗

bioRxiv2021

Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

systems biology↗

bioRxiv2021

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

biochemistry↗