Biology subjects

Chapman, E. A.

Publications and source records attributed to Chapman, E. A..

11 recordsLinked to original sources

bioRxiv2026

Extending the seasons at both ends? Understanding the physiological and genetic context required for stay green mediated yield increase in wheat (Triticum aestivum)

Flowering time and monocarpic senescence are tightly environmentally and genetically controlled. Typically, early flowering and staygreen traits are associated with opposing life-history strategies; stress avoidance versus adaptation; with flowering time an overarching regulator of crop cycle length. We developed RIL populations segregating for Ppd-1 and NAM-1 variation, which are otherwise isogenic. Multi-year field experiments enabled exploration and uncoupling of the relationship between heading and staygreen traits. Heading date manipulation enabled introduction of staygreen traits to their target breeding environments, characterised by a hot-finish. Under moderate stress, we report a 2.9% and 1.9% increase in grain width (P<0.0001), and 5.8% and 3.7% increase in TGW (P<0.0001), plus significantly greater yield (P<0.1) for late heading staygreen RILs homozygous for NAM-A1, and NAM-D1 missense variants, respectively. Grain yield increases were proportionate to the delay in senescence, being greater for the NAM-A1 than the NAM-D1 variant. For RIL populations segregating for both traits, senescence variation was observed relative to heading-date. Regarding grain yield, the staygreen trait-associated increase in source size could not compensate for the Ppd-1a associated pleiotropic reduction in sink size, even under hypothesised continental target breeding environments, with trait competition identified. Therefore, to maximise the benefits associated with staygreen traits, especially in early-heading favouring environments required targeted manipulation of source-sink dynamics, and we propose multiple strategies. HighlightStaygreen traits were associated with extending grain fill duration, increasing grain width, TGW and grain yield. There appears an antagonist relationship between earlier heading and staygreen traits.

plant biology↗

bioRxiv2025

Extracellular Matrix Alterations in Chronic Ischemic Cardiomyopathy Revealed by Quantitative Proteomics

Ischemic cardiomyopathy (ICM) is a leading cause of heart failure characterized by extensive remodeling of the cardiac extracellular matrix (ECM). While initially adaptive, ECM deposition following ischemic injury eventually turns maladaptive, promoting adverse cardiac remodeling. The strong link between the extent of fibrosis and adverse clinical outcomes has led to growing interest in ECM targeted therapies to prevent or reverse maladaptive cardiac remodeling in ICM; yet, the precise composition of the ECM in ICM remains poorly defined. In this study, we employed a sequential protein extraction enabled by the photocleavable surfactant Azo to enrich ECM proteins from left ventricular tissues of patients with end-stage ICM (n=16) and nonfailing donor hearts (n=16). High-resolution mass spectrometry-based quantitative proteomics identified and quantified over 6,000 unique protein groups, including 315 ECM proteins. We discovered significant upregulation of key ECM components, particularly glycoproteins, proteoglycans, collagens, and ECM regulators. Notably, LOXL1, FBLN1, and VCAN were among the most differentially expressed. Functional enrichment analyses revealed enhanced TGF{beta} signaling, integrin-mediated adhesion, and complement activation in ICM tissues, suggesting a feedback loop driving continued ECM deposition in the end-stage failing heart. Together, our findings provide a comprehensive proteomic landscape of ECM alterations in the end-stage ICM myocardium and identify promising molecular targets for therapeutic intervention.

biochemistry↗

bioRxiv2025

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

biochemistry↗

bioRxiv2025

Native Top-Down Proteomics of Endogenous Protein Complexes Enabled by Online Two-Dimensional Liquid Chromatography

Protein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes. The automated, online interfacing of size-exclusion and mixed-bed ion-exchange chromatography achieves high coverage of endogenous protein complexes. We further develop a multistage nTDMS approach that enables comprehensive structural characterization within the chromatographic timescale, capturing intact non-covalent complexes, released subunits/cofactors, and backbone fragments. Our analysis detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in less than two hours. Such technological leaps in high-throughput structural characterization of endogenous protein complexes will advance large-scale nTDP studies in health and disease.

biochemistry↗

bioRxiv2024

In-depth Characterization of S-Glutathionylation in Ventricular Myosin Light Chain 1 Across Species by Top-Down Proteomics

S-glutathionylation (SSG) is increasingly recognized as a critical signaling mechanism in the heart, yet SSG modifications in cardiac sarcomeric proteins remain understudied. Here we identified SSG of the ventricular isoform of myosin light chain 1 (MLC-1v) in human, swine, and mouse cardiac tissues using top-down mass spectrometry (MS)-based proteomics. Our results enabled the accurate identification, quantification, and site-specific localization of SSG in MLC-1v across different species. Notably, the endogenous SSG of MLC-1v was observed in human and swine cardiac tissues but not in mice. Treating non-reduced cardiac tissue lysates with GSSG elevated MLC-1v SSG levels across all three species.

biochemistry↗

bioRxiv2023

Integrated Proteomics Identifies Troponin I Isoform Switch as a Regulator of a Sarcomere-Metabolism Axis During Cardiac Regeneration

Adult mammalian cardiomyocytes have limited proliferative potential, and after myocardial infarction (MI), injured cardiac tissue is replaced with fibrotic scar rather than with functioning myocardium. In contrast, the neonatal mouse heart possesses a regenerative capacity governed by cardiomyocyte proliferation; however, a metabolic switch from glycolysis to fatty acid oxidation during postnatal development results in loss of this regenerative capacity. Interestingly, a sarcomere isoform switch also takes place during postnatal development where slow skeletal troponin I (ssTnI) is replaced with cardiac troponin I (cTnI). In this study, we first employ integrated quantitative bottom-up and top-down proteomics to comprehensively define the proteomic and sarcomeric landscape during postnatal heart maturation. Utilizing a cardiomyocyte-specific ssTnI transgenic mouse model, we found that ssTnI overexpression increased cardiomyocyte proliferation and the cardiac regenerative capacity of the postnatal heart following MI compared to control mice by histological analysis. Our global proteomic analysis of ssTnI transgenic mice following MI reveals that ssTnI overexpression induces a significant shift in the cardiac proteomic landscape. This shift is characterized by an upregulation of key proteins involved in glycolytic metabolism. Collectively, our data suggest that the postnatal TnI isoform switch may play a role in the metabolic shift from glycolysis to fatty acid oxidation during postnatal maturation. This underscores the significance of a sarcomere-metabolism axis during cardiomyocyte proliferation and heart regeneration.

developmental biology↗

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

Top-down Proteomics of Myosin Light Chain Isoforms Define Chamber-Specific Expression in the Human Heart

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, 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 and MLC-2a 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. Overall, top-down proteomics allowed us an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

systems biology↗

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↗

bioRxiv2020

Capturing and Selecting Senescence Variation in Wheat

Senescence is a highly quantitative trait, but in wheat the genetics underpinning senescence regulation remain relatively unknown. To select senescence variation, and ultimately identify novel genetic regulators, accurate characterisation of senescence phenotypes is essential. When investigating senescence, phenotyping efforts often focus on, or are limited to, visual assessment of the flag leaves. However, senescence is a whole plant process, involving remobilisation and translocation of resources into the developing grain. Furthermore, the temporal progression of senescence poses challenges regarding trait quantification and description, whereupon the different models and approaches applied result in varying definitions of apparently similar metrics. To gain a holistic understanding of senescence we phenotyped flag leaf and peduncle senescence progression, alongside grain maturation. Reviewing the literature, we identified techniques commonly applied in quantification of senescence variation and developed simple methods to calculate descriptive and discriminatory metrics. To capture senescence dynamism, we developed the idea of calculating thermal time to different flag leaf senescence scores, for which between year Spearmans rank correlations of r [≥] 0.59, P < 4.7 x 10-5(TT70), identify as an accurate phenotyping method. Following our experience of senescence trait genetic mapping, we recognised the need for singular metrics capable of discriminating senescence variation, identifying Thermal Time to Flag Leaf Senescence score of 70 (TT70) and Mean Peduncle senescence (MeanPed) scores as most informative. Moreover, grain maturity assessments confirmed a previous association between our staygreen traits and grain fill extension, illustrating trait functionality. Here we review different senescence phenotyping approaches and share our experiences of phenotyping two independent RIL populations segregating for staygreen traits. Together, we direct readers towards senescence phenotyping methods we found most effective, encouraging their use when investigating and discriminating senescence variation of differing genetic bases, and to aid trait selection and weighting in breeding and research programs alike.

plant biology↗

bioRxiv2020

Delaying or Delivering: Identification of novel NAM-1 alleles which delay senescence to extend grain fill duration of wheat

Senescence is a complex quantitative trait under genetic and environmental control, involving the remobilisation of resources from vegetative tissue into grain. Delayed senescence, or staygreen traits, are associated with conferring stress tolerance, with extended photosynthetic activity hypothesised to sustain grain filling. The genetics of senescence regulation are largely unknown, with senescence variation often correlated with phenological traits. Here, we confirm staygreen phenotypes of two Triticum aestivum cv. Paragon EMS mutants previously identified during a forward genetic screen and selected for their agronomic performance, similar phenology and differential senescence phenotypes. Through grain filling experiments, we confirm a positive relationship between onset of senescence and grain fill duration, reporting an associated [~]14 % increase in final dry grain weight for one mutant, P < 0.05. Recombinant Inbred Line (RIL) populations segregating for senescence were developed for trait mapping purposes, and phenotyped over multiple years under field conditions. Staygreen traits were mapped using exome-capture enabled bulk segregant analysis (BSA), whereupon senescence was quantified, and metrics compared to qualify senescence traits and aid RIL selection. Using BSA we mapped our two staygreen traits to two independent, dominant, loci of 4.8 and 16.7 Mb in size encompassing 56 and 142 genes. Combining single marker association analysis with variant effect prediction, we identified SNPs encoding self-validating mutations located in NAM-1 homoeologues and propose these as gene candidates. SummaryApplying an exome-capture enabled Bulk Segregant Analysis, we identified novel NAM-1 alleles underpinning two staygreen traits associated with enhancing grain fill duration and grain weight

plant biology↗