David S. Roberts

All Publications

• Directed evolution of genetically encoded LYTACs for cell-mediated delivery. Proceedings of the National Academy of Sciences of the United States of America Yang, J. L., Yamada-Hunter, S. A., Labanieh, L., Sotillo, E., Cheah, J. S., Roberts, D. S., Mackall, C. L., Bertozzi, C. R., Ting, A. Y. 2024; 121 (13): e2320053121

  Abstract

  Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.

  View details for DOI 10.1073/pnas.2320053121

  View details for PubMedID 38513100

• Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate. Journal of the American Society for Mass Spectrometry Chapman, E. A., Li, B. H., Krichel, B., Chan, H., Buck, K. M., Roberts, D. S., Ge, Y. 2024

  Abstract

  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 (alpha-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.

  View details for DOI 10.1021/jasms.3c00430

  View details for PubMedID 38422011

• A Lectin-Drug Conjugate CRISPR Screen Identifies Sortilin as the Lysosomal Trafficking Receptor for Galectin-1 Donnelly, J., Kamber, R., Wisnovsky, S., Roberts, D., Peltan, E., Bassik, M. OXFORD UNIV PRESS INC. 2023: 1055

  View details for Web of Science ID 001182824100216

• Identification of TAG-72 carriers and glycoproteomic mapping of the epitope Peltan, E. L., Roberts, D. S., Riley, N. M., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2023: 1047

  View details for Web of Science ID 001182824100198

• Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics. Nature communications Chapman, E. A., Roberts, D. S., Tiambeng, T. N., Andrews, J., Wang, M. D., Reasoner, E. A., Melby, J. A., Li, B. H., Kim, D., Alpert, A. J., Jin, S., Ge, Y. 2023; 14 (1): 8400

  Abstract

  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.

  View details for DOI 10.1038/s41467-023-43321-z

  View details for PubMedID 38110393

  View details for PubMedCentralID PMC10728164

• Mass Spectrometry-Based Multiomics Identifies Metabolic Signatures of Sarcopenia in Rhesus Monkey Skeletal Muscle. Journal of proteome research Pergande, M. R., Osterbauer, K. J., Buck, K. M., Roberts, D. S., Wood, N. N., Balasubramanian, P., Mann, M. W., Rossler, K. J., Diffee, G. M., Colman, R. J., Anderson, R. M., Ge, Y. 2023

  Abstract

  Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in "omics" technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offers new insights into the molecular mechanisms underlying sarcopenia for the evaluation and monitoring of a therapeutic treatment of sarcopenia.

  View details for DOI 10.1021/acs.jproteome.3c00474

  View details for PubMedID 37991985

• Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery. bioRxiv : the preprint server for biology Yang, J. L., Yamada-Hunter, S. A., Labanieh, L., Sotillo, E., Cheah, J. S., Roberts, D. S., Mackall, C. L., Ting, A. Y., Bertozzi, C. R. 2023

  Abstract

  Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation.

  View details for DOI 10.1101/2023.11.14.567117

  View details for PubMedID 38014030

  View details for PubMedCentralID PMC10680704

• Galectin-3 does not interact with RNA directly. Glycobiology Peltan, E. L., Riley, N. M., Flynn, R. A., Roberts, D. S., Bertozzi, C. R. 2023

  Abstract

  Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.

  View details for DOI 10.1093/glycob/cwad076

  View details for PubMedID 37815932

• Comprehensive Characterization of Endogenous Phospholamban Proteoforms Enabled by Photocleavable Surfactant and Top-down Proteomics. Analytical chemistry Rogers, H. T., Roberts, D. S., Larson, E. J., Melby, J. A., Rossler, K. J., Carr, A. V., Brown, K. A., Ge, Y. 2023

  Abstract

  Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modifications (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 method on a quadrupole time-of-flight MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance 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. Moreover, we applied our method to characterize PLN in disease and reported the significant reduction of PLN phosphorylation in human failing hearts with ischemic cardiomyopathy. 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.

  View details for DOI 10.1021/acs.analchem.3c01618

  View details for PubMedID 37607050

• Structure and dynamics of endogenous protein complexes in human heart tissue captured by native nanoproteomics. Research square Chapman, E. A., Roberts, D. S., Tiambeng, T. N., Andrews, J., Wang, M. D., Reasoner, E. A., Melby, J. A., Li, B. H., Kim, D., Alpert, A. J., Jin, S., Ge, Y. 2023

  Abstract

  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.

  View details for DOI 10.21203/rs.3.rs-3108087/v1

  View details for PubMedID 37461709

  View details for PubMedCentralID PMC10350235

• Structure and dynamics of endogenous protein complexes in human heart tissue captured by native nanoproteomics. bioRxiv : the preprint server for biology Chapman, E. A., Roberts, D. S., Tiambeng, T. N., Andrews, J., Wang, M., Reasoner, E. A., Melby, J. A., Li, B. H., Kim, D., Alpert, A. J., Jin, S., Ge, Y. 2023

  Abstract

  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.

  View details for DOI 10.1101/2023.06.13.544817

  View details for PubMedID 37398031

• Bromodomain-containing Protein 4 regulates innate inflammation via modulation of alternative splicing. Frontiers in immunology Mann, M. W., Fu, Y., Gearhart, R. L., Xu, X., Roberts, D. S., Li, Y., Zhou, J., Ge, Y., Brasier, A. R. 2023; 14: 1212770

  Abstract

  Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In the context of airway viral infection, BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream epithelial plasticity. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not well understood. Given BRD4's interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing.To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep and integrated coverage of the proteomic and transcriptomic landscapes of human small airway epithelial cells exposed to viral challenge and treated with BRD4i.We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 (IFRD1) and X-Box Binding Protein 1 (XBP1), related to the innate immune response and the unfolded protein response (UPR). We identify requirement of BRD4 for expression of serine-arginine splicing factors, splicosome components and the Inositol-Requiring Enzyme 1 IREα affecting immediate early innate response and the UPR.These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing via modulating splicing factor expression in virus-induced innate signaling.

  View details for DOI 10.3389/fimmu.2023.1212770

  View details for PubMedID 37435059

  View details for PubMedCentralID PMC10331468

• Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart. Journal of molecular and cellular cardiology Bayne, E. F., Rossler, K. J., Gregorich, Z. R., Aballo, T. J., Roberts, D. S., Chapman, E. A., Guo, W., Palecek, S. P., Ralphe, J. C., Kamp, T. J., Ge, Y. 2023

  Abstract

  Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an 'atrial' and 'ventricular' isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

  View details for DOI 10.1016/j.yjmcc.2023.06.003

  View details for PubMedID 37327991

• MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing. Bioinformatics (Oxford, England) Larson, E. J., Pergande, M. R., Moss, M. E., Rossler, K. J., Wenger, R. K., Krichel, B., Josyer, H., Melby, J. A., Roberts, D. S., Pike, K., Shi, Z., Chan, H. J., Knight, B., Rogers, H. T., Brown, K. A., Ong, I. M., Jeong, K., Marty, M., McIlwain, S. J., Ge, Y. 2023

  Abstract

  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.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/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.Supplementary data are available at Bioinformatics online.

  View details for DOI 10.1093/bioinformatics/btad359

  View details for PubMedID 37294807

• High sensitivity top-down proteomics captures single muscle cell heterogeneity in large proteoforms. Proceedings of the National Academy of Sciences of the United States of America Melby, J. A., Brown, K. A., Gregorich, Z. R., Roberts, D. S., Chapman, E. A., Ehlers, L. E., Gao, Z., Larson, E. J., Jin, Y., Lopez, J. R., Hartung, J., Zhu, Y., McIlwain, S. J., Wang, D., Guo, W., Diffee, G. M., Ge, Y. 2023; 120 (19): e2222081120

  Abstract

  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 detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals 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.

  View details for DOI 10.1073/pnas.2222081120

  View details for PubMedID 37126723

  View details for PubMedCentralID PMC10175728

• Integrated proteomics reveals alterations in sarcomere composition and developmental processes during postnatal swine heart development JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Aballo, T. J., Roberts, D. S., Bayne, E. F., Zhu, W., Walcott, G., Mahmoud, A. I., Zhang, J., Ge, Y. 2023; 176: 33-40

  Abstract

  The neonatal swine heart possesses an endogenous ability to regenerate injured myocardium through the proliferation of pre-existing cardiomyocyte (CM) populations. However, this regenerative capacity is lost shortly after birth. Normal postnatal developmental processes and the regenerative capacity of mammalian hearts are tightly linked, but not much is known about how the swine cardiac proteome changes throughout postnatal development. Herein, we integrated robust and quantitative targeted "top-down" and global "bottom-up" proteomic workflows to comprehensively define the dynamic landscape of the swine cardiac proteome throughout postnatal maturation. Using targeted top-down proteomics, we were able to identify significant alterations in sarcomere composition, providing new insight into the proteoform landscape of sarcomeres that can disassemble, a process necessary for productive CM proliferation. Furthermore, we quantified global changes in protein abundance using bottom-up proteomics, identified over 700 differentially expressed proteins throughout postnatal development, and mapped these proteins to changes in developmental and metabolic processes. We envision these results will help guide future investigations to comprehensively understand endogenous cardiac regeneration toward the development of novel therapeutic strategies for heart failure.

  View details for DOI 10.1016/j.yjmcc.2023.01.004

  View details for Web of Science ID 000963589200001

  View details for PubMedID 36657638

  View details for PubMedCentralID PMC10006350

• Bromodomain-containing Protein 4 Regulates Innate Inflammation in Airway Epithelial Cells via Modulation of Alternative Splicing. bioRxiv : the preprint server for biology Mann, M., Fu, Y., Xu, X., Roberts, D. S., Li, Y., Zhou, J., Ge, Y., Brasier, A. R. 2023

  Abstract

  Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In airway viral infection, non-toxic BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream remodeling. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not as well understood. Based on its interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing. To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep and integrated coverage of the proteomic and transcriptomic landscapes of human small airway epithelial cells exposed to viral challenge and treated with BRD4i. The transcript-level data was further interrogated for alternative splicing analysis, and the resulting data sets were correlated to identify pathways subject to post-transcriptional regulation. We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 ( IFRD1 ) and X-Box Binding Protein 1 ( XBP1 ), related to the innate immune response and the unfolded protein response, respectively. These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing in innate signaling.

  View details for DOI 10.1101/2023.01.17.524257

  View details for PubMedID 36711789

  View details for PubMedCentralID PMC9882210

• Synthesis, Self-Assembly Properties, and Degradation Characterization of a Nonionic Photocleavable Azo-Sulfide Surfactant Family LANGMUIR Brown, K. A., Gugger, M. K., Roberts, D. S., Moreno, D., Chae, P., Ge, Y., Jin, S. 2023: 1465-1473

  Abstract

  We report the synthesis and characterization of a new family of maltose-derived nonionic surfactants that contain a photocleavable azo-sulfide linker (mAzo). The self-assembly properties of these surfactants were investigated using surface tension measurements to determine the critical micelle concentration (CMC), dynamic light scattering (DLS) to reveal the hydrodynamic radius of their self-assemblies, and transmission electron microscopy (TEM) to elucidate the micelle morphology. Ultraviolet-visible (UV-visible) spectroscopy confirmed the rapid photodegradation of these surfactants, but surface tension measurements of the surfactant solutions before and after degradation showed unusual degradation products. The photodegradation process was further studied using online liquid chromatography coupled with mass spectrometry (LC-MS),which revealed that these surfactants can form another photo-stable surfactant post-degradation. Finally, traditionally challenging proteins from heart tissue were solubilized using the mAzo surfactants to demonstrate their potential in biological applications.

  View details for DOI 10.1021/acs.langmuir.2c02820

  View details for Web of Science ID 000920312000001

  View details for PubMedID 36638323

  View details for PubMedCentralID PMC10164600

• Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL JOURNAL OF EXTRACELLULAR VESICLES Gupta, K., Brown, K. A., Hsieh, M. L., Hoover, B. M., Wang, J., Khoury, M. K., Pilli, V., Beyer, R. H., Voruganti, N. R., Chaudhary, S., Roberts, D. S., Murphy, R. M., Hong, S., Ge, Y., Liu, B. 2022; 11 (9): e12261

  Abstract

  Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase-3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

  View details for DOI 10.1002/jev2.12261

  View details for Web of Science ID 000849847400001

  View details for PubMedID 36063142

  View details for PubMedCentralID PMC9443950

• Distinct core glycan and O-glycoform utilization of SARS-CoV-2 Omicron variant Spike protein RBD revealed by top-down mass spectrometry CHEMICAL SCIENCE Roberts, D. S., Mann, M., Li, B. H., Kim, D., Braiser, A. R., Jin, S., Ge, Y. 2022; 13 (36): 10944-10949

  Abstract

  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.

  View details for DOI 10.1039/d2sc02132c

  View details for Web of Science ID 000850555700001

  View details for PubMedID 36320702

  View details for PubMedCentralID PMC9491206

• One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant ANALYTICAL CHEMISTRY Buck, K. M., Roberts, D. S., Aballo, T. J., Inman, D. R., Jin, S., Ponik, S., Brown, K. A., Ge, Y. 2022; 94 (20): 7164-7168

  Abstract

  Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with extremely high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content in exosomes, but the current methods require laborious and time-consuming multistep sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, to simplify exosomal lysis, effectively extract proteins, and expedite digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3466 proteins and quantified 2288 proteins using a reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. Here, 3166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision that this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.

  View details for DOI 10.1021/acs.analchem.2c01252

  View details for Web of Science ID 000886783000001

  View details for PubMedID 35543580

  View details for PubMedCentralID PMC9297302

• Sustainable Coproduction of Two Disinfectants via Hydroxide-Balanced Modular Electrochemical Synthesis Using a Redox Reservoir ACS CENTRAL SCIENCE Wang, R., Sheng, H., Wang, F., Li, W., Roberts, D. S., Jin, S. 2021; 7 (12): 2083-2091

  Abstract

  Challenges posed by the sacrificial auxiliary reactions and expensive ion-exchange membranes in conventional electrosynthesis necessitate developing new electrochemical processes to enable efficient and sustainable distributed electrochemical manufacturing. Modular electrochemical synthesis (ModES) using a redox reservoir (RR) offers a promising membrane-free approach to improve energy efficiency and reduce waste through the pairing of multiple independent oxidative and reductive half-reactions; however, undesired ion-imbalance and induced pH changes in the existing ModES process limit sustained production. Here we present Ni(OH)2 as a heterogeneous RR that can selectively store and transport the hydroxide ions involved in the target half-reactions by reversible conversion with NiOOH to enable an ion-balanced ModES of two common disinfectants, hydrogen peroxide (H2O2) and sodium hypochlorite (NaClO). This hydroxide-balanced ModES realizes stable operation without appreciable pH swing to accumulate practically useful concentrations of H2O2 and NaClO up to 251 and 481 ppm, respectively. These results illustrate additional design principles for electrosynthesis without sacrificial auxiliary reactions and the need for ion-selective RRs for modular electrochemical manufacturing.

  View details for DOI 10.1021/acscentsci.1c01157

  View details for Web of Science ID 000744186200016

  View details for PubMedID 34963900

  View details for PubMedCentralID PMC8704031

• Multiomics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem-Cell-Derived Cardiomyocytes JOURNAL OF PROTEOME RESEARCH Bayne, E. F., Simmons, A. D., Roberts, D. S., Zhu, Y., Aballo, T. J., Wancewicz, B., Palecek, S. P., Ge, Y. 2021; 20 (10): 4646-4654

  Abstract

  Human pluripotent stem-cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, thus far, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo. To gain global insight into hPSC-CM biology, we established a multiomics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multidimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures and analyzed these extracts using high-resolution mass spectrometry. Using this method, we annotated 205 metabolites/lipids and 4319 proteins from 106 cells with high reproducibility. We further integrated the proteome and metabolome measurements to create network profiles of molecular phenotypes for hPSC-CMs. Out of 310 pathways identified using metabolomics and proteomics, 40 pathways were considered significantly overrepresented (false-discovery-rate-corrected p ≤ 0.05). Highly populated pathways included those involved in protein synthesis (ribosome, spliceosome), ATP generation (oxidative phosphorylation), and cardiac muscle contraction. This multiomics method achieves a deep coverage of metabolites and proteins, creating a multidimensional view of the hPSC-CM phenotype, which provides a strong technological foundation to advance the understanding of hPSC-CM biology. Raw data are available in the MassIVE repository with identifier MSV000088010.

  View details for DOI 10.1021/acs.jproteome.1c00611

  View details for Web of Science ID 000704711700004

  View details for PubMedID 34499502

  View details for PubMedCentralID PMC8714023

• Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Roberts, D. S., Mann, M., Melby, J. A., Larson, E. J., Zhu, Y., Brasier, A. R., Jin, S., Ge, Y. 2021; 143 (31): 12014-12024

  Abstract

  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 plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity 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 by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach 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.

  View details for DOI 10.1021/jacs.1c02713

  View details for Web of Science ID 000684581100019

  View details for PubMedID 34328324

  View details for PubMedCentralID PMC8353889

• High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry ANALYTICAL CHEMISTRY Larson, E. J., Roberts, D. S., Melby, J. A., Buck, K. M., Zhu, Y., Zhou, S., Han, L., Zhang, Q., Ge, Y. 2021; 93 (29): 10013-10021

  Abstract

  Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-to-antibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by low-energy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.

  View details for DOI 10.1021/acs.analchem.1c00150

  View details for Web of Science ID 000679366000009

  View details for PubMedID 34258999

  View details for PubMedCentralID PMC8319120

• Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro JOURNAL OF PROTEOME RESEARCH Aballo, T. J., Roberts, D. S., Melby, J. A., Buck, K. M., Brown, K. A., Ge, Y. 2021; 20 (8): 4203-4211

  Abstract

  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 cleanup 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 4000 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.

  View details for DOI 10.1021/acs.jproteome.1c00446

  View details for Web of Science ID 000684095100038

  View details for PubMedID 34236868

  View details for PubMedCentralID PMC8349881

• Novel Strategies to Address the Challenges in Top-Down Proteomics JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Melby, J. A., Roberts, D. S., Larson, E. J., Brown, K. A., Bayne, E. F., Jin, S., Ge, Y. 2021; 32 (6): 1278-1294

  Abstract

  Top-down mass spectrometry (MS)-based proteomics is a powerful technology for comprehensively characterizing proteoforms to decipher post-translational modifications (PTMs) together with genetic variations and alternative splicing isoforms toward a proteome-wide understanding of protein functions. In the past decade, top-down proteomics has experienced rapid growth benefiting from groundbreaking technological advances, which have begun to reveal the potential of top-down proteomics for understanding basic biological functions, unraveling disease mechanisms, and discovering new biomarkers. However, many challenges remain to be comprehensively addressed. In this Account & Perspective, we discuss the major challenges currently facing the top-down proteomics field, particularly in protein solubility, proteome dynamic range, proteome complexity, data analysis, proteoform-function relationship, and analytical throughput for precision medicine. We specifically review the major technology developments addressing these challenges with an emphasis on our research group's efforts, including the development of top-down MS-compatible surfactants for protein solubilization, functionalized nanoparticles for the enrichment of low-abundance proteoforms, strategies for multidimensional chromatography separation of proteins, and a new comprehensive user-friendly software package for top-down proteomics. We have also made efforts to connect proteoforms with biological functions and provide our visions on what the future holds for top-down proteomics.

  View details for DOI 10.1021/jasms.1c00099

  View details for Web of Science ID 000714641000002

  View details for PubMedID 33983025

  View details for PubMedCentralID PMC8310706

• Discovery of RSV-Induced BRD4 Protein Interactions Using Native Immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) Mass Spectrometry VIRUSES-BASEL Mann, M., Roberts, D. S., Zhu, Y., Li, Y., Zhou, J., Ge, Y., Brasier, A. R. 2021; 13 (3)

  Abstract

  Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4's acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

  View details for DOI 10.3390/v13030454

  View details for Web of Science ID 000634215000001

  View details for PubMedID 33799525

  View details for PubMedCentralID PMC8000986

• Functionally Integrated Top-Down Proteomics for Standardized Assessment of Human Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues JOURNAL OF PROTEOME RESEARCH Melby, J. A., de Lange, W. J., Zhang, J., Roberts, D. S., Mitchell, S. D., Tucholski, T., Kim, G., Kyrvasilis, A., McIlwain, S. J., Kamp, T. J., Ralphe, J., Ge, Y. 2021; 20 (2): 1424-1433

  Abstract

  Three-dimensional (3D) human induced pluripotent stem cell-derived engineered cardiac tissues (hiPSC-ECTs) have emerged as a promising alternative to two-dimensional hiPSC-cardiomyocyte monolayer systems because hiPSC-ECTs are a closer representation of endogenous cardiac tissues and more faithfully reflect the relevant cardiac pathophysiology. The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.

  View details for DOI 10.1021/acs.jproteome.0c00830

  View details for Web of Science ID 000618540700026

  View details for PubMedID 33395532

  View details for PubMedCentralID PMC7872211

• Top-down proteomics: challenges, innovations, and applications in basic and clinical research EXPERT REVIEW OF PROTEOMICS Brown, K. A., Melby, J. A., Roberts, D. S., Ge, Y. 2020; 17 (10): 719-733

  Abstract

  Introduction- A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics toward precision medicine. However, many challenges remain to unravel the complex nature of diseases. Areas covered- Changes in protein isoform expression and post-translation modifications (PTMs) have gained recognition for their role in underlying disease mechanisms. Top-down mass spectrometry (MS)-based proteomics is increasingly recognized as an important method for the comprehensive characterization of proteoforms that arise from alternative splicing events and/or PTMs for basic and clinical research. Here, we review the challenges, technological innovations, and recent studies that utilize top-down proteomics to elucidate changes in the proteome with an emphasis on its use to study heart diseases. Expert opinion- Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development . Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs. Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.

  View details for DOI 10.1080/14789450.2020.1855982

  View details for Web of Science ID 000599689100001

  View details for PubMedID 33232185

  View details for PubMedCentralID PMC7864889

• MASH Explorer: A Universal Software Environment for Top-Down Proteomics JOURNAL OF PROTEOME RESEARCH Wu, Z., Roberts, D. S., Melby, J. A., Wenger, K., Wetzel, M., Gu, Y., Ramanathan, S., Bayne, E. F., Liu, X., Sun, R., Ong, I. M., McIlwain, S. J., Ge, Y. 2020; 19 (9): 3867-3876

  Abstract

  Top-down mass spectrometry (MS)-based proteomics enable a comprehensive analysis of proteoforms with molecular specificity to achieve a proteome-wide understanding of protein functions. However, the lack of a universal software for top-down proteomics is becoming increasingly recognized as a major barrier, especially for newcomers. Here, we have developed MASH Explorer, a universal, comprehensive, and user-friendly software environment for top-down proteomics. MASH Explorer integrates multiple spectral deconvolution and database search algorithms into a single, universal platform which can process top-down proteomics data from various vendor formats, for the first time. It addresses the urgent need in the rapidly growing top-down proteomics community and is freely available to all users worldwide. With the critical need and tremendous support from the community, we envision that this MASH Explorer software package will play an integral role in advancing top-down proteomics to realize its full potential for biomedical research.

  View details for DOI 10.1021/acs.jproteome.0c00469

  View details for Web of Science ID 000569378700026

  View details for PubMedID 32786689

  View details for PubMedCentralID PMC7728713

• Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum NATURE COMMUNICATIONS Tiambeng, T. N., Roberts, D. S., Brown, K. A., Zhu, Y., Chen, B., Wu, Z., Mitchell, S. D., Guardado-Alvarez, T. M., Jin, S., Ge, Y. 2020; 11 (1): 3903

  Abstract

  Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>1010 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.

  View details for DOI 10.1038/s41467-020-17643-1

  View details for Web of Science ID 000561120500002

  View details for PubMedID 32764543

  View details for PubMedCentralID PMC7411019

• Quantum Ensembles of Silicon Nanoparticles: Discrimination of Static and Dynamic Photoluminescence Quenching Processes JOURNAL OF PHYSICAL CHEMISTRY C Hollett, G., Roberts, D. S., Sewell, M., Wensley, E., Wagner, J., Murray, W., Krotz, A., Toth, B., Vijayakumar, V., Sailor, M. J. 2019; 123 (29): 17976-17986

  Abstract

  Porous silicon photoluminescence is characterized by a broad emission band that displays unusually long (tens to hundreds of micro-seconds), wavelength-dependent emissive lifetimes. The photoluminescence is associated with quantum confinement of excitons in silicon nanocrystallites contained within the porous matrix, and the broad emission spectrum derives from the wide distribution of nanocrystallite sizes in the material. The longer emissive lifetimes in the ensemble of quantum-confined emitters correspond to the larger nanocrystallites, with their longer wavelengths of emission. The quenching of this photoluminescence by aromatic, redox-active molecules aminochrome (AMC), dopamine, adrenochrome, sodium anthraquinone-2-sulfonate, benzyl viologen dichloride, methyl viologen dichloride hydrate, and ethyl viologen dibromide is studied, and dynamic and static quenching mechanisms are distinguished by the emission lifetime analysis. Because of the dependence of the emission lifetime on emission wavelength from the silicon nanocrystallite ensemble, a pronounced blue shift is observed in the steady-state emission spectrum upon exposure to dynamic-type quenchers. Conversely, static-type quenching systems show uniform quenching across all emission wavelengths. Thus, the difference between static and dynamic quenching mechanisms is readily distinguished by ratiometric photoluminescence spectroscopy. The application of this concept to imaging of AMC, the oxidized form of the neurotransmitter dopamine that is of interest for its role in neurodegenerative diseases, is demonstrated. It is found that static electron acceptors result in no ratiometric contrast, while AMC shows a strong contrast, allowing ready visualization in a 2-D imaging experiment.

  View details for DOI 10.1021/acs.jpcc.9b04334

  View details for Web of Science ID 000477785000037

  View details for PubMedID 32489514

  View details for PubMedCentralID PMC7266134

• Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: Enrichment of the human heart phosphoproteome NANO RESEARCH Roberts, D. S., Chen, B., Tiambeng, T. N., Wu, Z., Ge, Y., Jin, S. 2019; 12 (6): 1473-1481

  Abstract

  A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications. After further coupling a phosphate-specific affinity ligand, these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture. Moreover, correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch. Furthermore, by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we were able to specifically enrich, identify, and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby enabling downstream proteomics applications.

  View details for DOI 10.1007/s12274-019-2418-4

  View details for Web of Science ID 000469405300032

  View details for PubMedID 31341559

  View details for PubMedCentralID PMC6656398

• Analysis of cardiac troponin proteoforms by top-down mass spectrometry POST-TRANSLATIONAL MODIFICATIONS THAT MODULATE ENZYME ACTIVITY Tiambeng, T. N., Tucholski, T., Wu, Z., Zhu, Y., Mitchell, S. D., Roberts, D. S., Jin, Y., Ge, Y., Garcia, B. A. 2019; 626: 347-374

  Abstract

  The cardiac troponin complex, composed of three regulatory proteins (cTnI, cTnT, TnC), functions as the critical regulator of cardiac muscle contraction and relaxation. Myofilament protein-protein interactions are regulated by post-translational modifications (PTMs) to the protein constituents of this complex. Dysregulation of troponin PTMs, particularly phosphorylation, results in altered cardiac contractility. Altered PTMs and isoforms have been increasingly recognized as the molecular mechanisms underlying heart diseases. Therefore, it is essential to comprehensively analyze cardiac troponin proteoforms that arise from PTMs, alternative splicing, and sequence variations. In this chapter, we described two detailed protocols for the enrichment and purification of endogenous cardiac troponin proteoforms from cardiac tissue. Subsequently, mass spectrometry (MS)-based top-down proteomics utilizing online liquid chromatography (LC)/quadrupole time-of-flight (Q-TOF) MS for separation, profiling, and quantification of the troponins was demonstrated. Characterization of troponin amino acid sequence and the localization of PTMs were shown using Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Furthermore, we described the use of MASH software, a comprehensive and free software package developed in our lab, for top-down proteomics data analysis. The methods we described can be applied for the analysis of troponin proteoforms in cardiac tissues, from animal models to human clinical samples, for heart disease.

  View details for DOI 10.1016/bs.mie.2019.07.029

  View details for Web of Science ID 000500385200016

  View details for PubMedID 31606082

  View details for PubMedCentralID PMC7557121

• Oriented Nanofibrous Polymer Scaffolds Containing Protein-Loaded Porous Silicon Generated by Spray Nebulization ADVANCED MATERIALS Zuidema, J. M., Kumeria, T., Kim, D., Kang, J., Wang, J., Hollett, G., Zhang, X., Roberts, D. S., Chan, N., Dowling, C., Blanco-Suarez, E., Allen, N. J., Tuszynski, M. H., Sailor, M. J. 2018; 30 (12): e1706785

  Abstract

  Oriented composite nanofibers consisting of porous silicon nanoparticles (pSiNPs) embedded in a polycaprolactone or poly(lactide-co-glycolide) matrix are prepared by spray nebulization from chloroform solutions using an airbrush. The nanofibers can be oriented by an appropriate positioning of the airbrush nozzle, and they can direct growth of neurites from rat dorsal root ganglion neurons. When loaded with the model protein lysozyme, the pSiNPs allow the generation of nanofiber scaffolds that carry and deliver the protein under physiologic conditions (phosphate-buffered saline (PBS), at 37 °C) for up to 60 d, retaining 75% of the enzymatic activity over this time period. The mass loading of protein in the pSiNPs is 36%, and in the resulting polymer/pSiNP scaffolds it is 3.6%. The use of pSiNPs that display intrinsic photoluminescence (from the quantum-confined Si nanostructure) allows the polymer/pSiNP composites to be definitively identified and tracked by time-gated photoluminescence imaging. The remarkable ability of the pSiNPs to protect the protein payload from denaturation, both during processing and for the duration of the long-term aqueous release study, establishes a model for the generation of biodegradable nanofiber scaffolds that can load and deliver sensitive biologics.

  View details for DOI 10.1002/adma.201706785

  View details for Web of Science ID 000428348000026

  View details for PubMedID 29363828

  View details for PubMedCentralID PMC6475500

• Preparation of Photoluminescent Porous Silicon Nanoparticles by High-Pressure Microfluidization PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION Roberts, D. S., Estrada, D., Yagi, N., Anglin, E. J., Chan, N. A., Sailor, M. J. 2017; 34 (3)

  View details for DOI 10.1002/ppsc.201600326

  View details for Web of Science ID 000398089100005