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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

OmicsMLMentor: A Web Application for Guided Machine Learning Analysis of Omics Data

Expression-based omics technologies (e.g. proteomics, metabolomics, transcriptomics, etc.) increasingly rely on supervised and unsupervised machine learning (ML) models to find key biomolecules distinguishing conditions, identify natural groupings in biological data, or generate predictions for outcomes of interest. Fitting ML models to omics data presents several challenges, including handling missing data, selecting a normalization method, choosing a valid model, and optimizing hyperparameters, all requiring statistical programming skills to address these challenges. Thus, the open-source web application SLOPE was designed to lower the barrier to ML modeling for omics data. SLOPE supports the fitting of 15 ML models (10 supervised and 5 unsupervised) tailored to omics datasets, such as proteomics, metabolomics, lipidomics, and transcriptomics. SLOPE offers several omics-specific features, including methods for handling missingness (imputation, conversion, removal), normalization tests, ranking of models based on the structure of a user’s data and user input, and optimal hyperparameter selections using cross-validation splits. By streamlining ML workflows for omics analysis, SLOPE address critical gaps in existing online web tools, facilitating a broader adoption of these models for omics research. Here, SLOPE is applied to data from a lignin exposure study to highlight the workflow for fitting both supervised and unsupervised models to data.

lipidomics↗
DOE OSTI2024

Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants

Abstract Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, one of the phenotypes of the CAX1 mutant being altered with tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1, and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here, we used synchrotron X-ray fluorescence (SXRF) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium and manganese. Proteomic analysis on isolated Arabidopsis trichomes showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

AI-accelerated protein-ligand docking for SARS-CoV-2 is 100-fold faster with no significant change in detection

Protein-ligand docking is a computational method for identifying drug leads. The method is capable of narrowing a vast library of compounds down to a tractable size for downstream simulation or experimental testing and is widely used in drug discovery. While there has been progress in accelerating scoring of compounds with artificial intelligence, few works have bridged these successes back to the virtual screening community in terms of utility and forward-looking development. We demonstrate the power of high-speed ML models by scoring 1 billion molecules in under a day (50 k predictions per GPU seconds). We showcase a workflow for docking utilizing surrogate AI-based models as a pre-filter to a standard docking workflow. Our workflow is ten times faster at screening a library of compounds than the standard technique, with an error rate less than 0.01% of detecting the underlying best scoring 0.1% of compounds. Our analysis of the speedup explains that another order of magnitude speedup must come from model accuracy rather than computing speed. In order to drive another order of magnitude of acceleration, we share a benchmark dataset consisting of 200 million 3D complex structures and 2D structure scores across a consistent set of 13 million “in-stock” molecules over 15 receptors, or binding sites, across the SARS-CoV-2 proteome. We believe this is strong evidence for the community to begin focusing on improving the accuracy of surrogate models to improve the ability to screen massive compound libraries 100 × or even 1000 × faster than current techniques and reduce missing top hits. The technique outlined aims to be a fast drop-in replacement for docking for screening billion-scale molecular libraries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure of the essential inner membrane lipopolysaccharide–PbgA complex

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Signatures of cysteine oxidation on muscle structural and contractile proteins are associated with physical performance and muscle function in older adults: Study of Muscle, Mobility and Aging (SOMMA)

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO 2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO 2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (R = 0.48) between a higher oxidation level of eight Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impairs muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Towards an Understanding of Oxidative Damage in an a-L-Arabinofuranosidase of Trichoderma reesei : a Molecular Dynamics Approach

Trichoderma reesei is a ‘workhorse’ fungus that produces glycosyl hydrolases (e.g., cellulases) at high titers for use in industrial bioprocessing. In this study, we focused on two side-chain hemicellulases, a-L-arabinofuranosidases and a-D-galactosidases, which are important for the treatment of lignocellulosic biomass, but susceptible to oxidative damage that can occur during industrial processing. The molecular details that render these enzymes inactive have not yet been identified. To approach this issue, we used proteomics to identify amino acid residues that were oxidized after a relevant oxidative treatment (Fenton reaction). These oxidative modifications were included in the 3D-protein structures, and using molecular dynamics simulations, we then studied the behaviors of non-modified and oxidized enzymes. These simulations showed significant alterations of the conformational stability of proteins when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized proteins. Finally, free energy landscape plots showed that there was a more rugged energy surface in the oxidized proteins, implying a less favorable reaction pathway. These results reveal the basis for loss of function in these carbohydrate active enzymes (CAZYs) in T. reesei.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of At ZNG1 , At MAP1A , At MAP2A , and At MAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. In conclusion, RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Endophyte-Promoted Phosphorus Solubilization in Populus

Phosphorus is an important plant nutrient, but in soils the majority of phosphorus, present in the form of a phosphate, forms metal complexes in soil making it relatively unavailable to plants. Therefore, inorganic phosphate solubilization is an important process of plant growth promotion by plant associated bacteria and fungi. Non nodulating plant species have been shown to thrive in low-nutrient environments, in some instances by relying on plant associated microorganisms called endophytes. These microorganisms live within the plant and help supply nutrients for the plant. Despite their potential enormous environmental importance, there are a limited number of studies looking at the direct molecular impact of phosphate solubilizing endophytic bacteria on the host plant. In this work, we studied the impact of two endophyte strains of wild poplar (Populus trichocarpa) that solubilize phosphate. Using a combination of x-ray imaging, spectroscopy methods, and proteomics we report direct evidence of endophyte-promoted phosphorus uptake in poplar. We found that the solubilized phosphate inside the plant may react and become insoluble again suggesting that the phosphate could be re-released within the plant by endophytes. Using synchrotron x-ray fluorescence spectromicroscopy combined with x-ray absorption near-edge structure, we visualized the nutrient phosphorus inside poplar roots inoculated by the selected endophytes and found the phosphorus in both forms of organic and inorganic phosphates inside the root. Tomography-based root imaging revealed a markedly different root biomass and root architecture for poplar samples inoculated with the phosphate solubilizing bacteria strains. Proteomics characterization on poplar roots coupled with protein network analysis revealed novel proteins and metabolic pathways with possible involvement in endophyte enriched phosphorus uptake. These findings suggest an important role of endophytes for phosphorus acquisition and provide a deeper understanding of the critical symbiotic associations between poplar and the endophytic bacteria.

36 MATERIALS SCIENCE↗
DOE OSTI2021

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain

Ca 2+ /calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the “PTP-dead” C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A giant virus infecting the amoeboflagellate Naegleria

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product’s primary structure reliably and reproducibly. The major objective of the present study was to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up approaches, such as confirming protein integrity, sequencing variable domains, ruling out artefacts, and revealing modifications and their locations. A panel of three mAbs was selected and centrally provided to twenty laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). A variety of different MS instrument platforms and ion dissociation techniques were employed. Overall, the present study confirms that MD/TD MS strategies are valuable and readily available tools in laboratories worldwide. They provide complementary information to the bottom-up approach and can add unique information, which may be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. One of the limitations revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS, but the number of TD/MD MS experts is still very low.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The plant endoplasmic reticulum: an organized chaos of tubules and sheets with multiple functions

The endoplasmic reticulum is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The endoplasmic reticulum structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review,we report on the findings that contributed towards the understanding of the endoplasmic reticulum morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Montage electron tomography of vitrified specimens

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2022

Harnessing Biomineralization Potential of Metal-Sequestering Bacteria for Rare Earth Elements (REE) Material Synthesis

Extremophilic methylotrophs, such as Methylotuvimicrobium alcaliphilum 20ZR are known for their natural ability to carry out REE capture and uptake. It has been predicted that proteins associated with surface layers facilitate transport and homeostasis of essential metals. Indeed, electron microscopic analysis coupled with antibody staining revealed that cell envelop of metal-grown M. alcaliphilum 20ZR contains metal-binding proteins in the base of the cup-shaped structures of S-layers. While less explored, the proteins associated with S-layers are also predicted to contribute to scavenging of other minerals essential for core metabolism. Here, we employed bottom-up proteomic study to compare the protein content of S-layer fractions of M. alcaliphilum 20ZR cultures grown in the presence and absence trace metals to identify putative components REE-scavenging machinery.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Implementation of Ion Mobility Spectrometry-Based Separations in Structures for Lossless Ion Manipulations (SLIM)

Structures for Lossless Ion Manipulations (SLIM) is a powerful variant of traveling wave ion mobility spectrometry (TW-IMS) that uses a serpentine pattern of microelectrodes deposited onto printed circuit boards to achieve ultralong ion path lengths (13.5 m). Ions are propelled through SLIM platforms via arrays of TW electrodes while RF and DC electrodes provide radial confinement, establishing near lossless transmission. The recent ability to cycle ions multiple times through a SLIM has allowed ion path lengths to exceed 1000 m, providing unprecedented separation power and the ability to observe ion structural conformations unobtainable with other IMS technologies. The combination of high separation power, high signal intensity, and the ability to couple with mass spectrometry places SLIM in the unique position of being able to address longstanding proteomics and metabolomics challenges by allowing the characterization of isomeric mixtures containing low abundance analytes.

Hollerbach, Adam L.↗
DOE OSTI2025

Sorted-cell proteomics reveals an AT1-associated epithelial cornification phenotype and suggests endothelial redox imbalance in human bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease characterized by inflammation and scarring leading to long-term tissue damage. Previous whole tissue proteomics identified BPD-specific proteome changes and cell type shifts. Little is known about the proteome-level changes within specific cell populations in disease. Here, we sorted epithelial (EPI) and endothelial (ENDO) cell populations based on their differential surface markers from normal and BPD human lungs. Using a low-input compatible sample preparation method (MicroPOT), proteins were extracted and digested into peptides and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis. Of the 4,970 proteins detected, 293 were modulated in abundance or detection in the EPI population and 422 were modulated in ENDO cells. Modulation of proteins associated with actin-cytoskeletal function, such as SCEL, LMO7, and TBA1B was observed in the BPD EPIs. Using confocal imaging and analysis, we validated the presence of aberrant multilayer-like structures comprising SCEL and LMO7, known to be associated with epidermal cornification, in the human BPD lung. This is the first report of the accumulation of cornification-associated proteins in BPD. Their localization in the alveolar parenchyma, primarily associated with alveolar type 1 (AT1) cells, suggests a role in the BPD postinjury response. In the ENDOs, redox balance and mitochondrial function pathways were modulated. Alternative mRNA splicing and cell proliferative functions were elevated in both populations, suggesting potential dysregulation of cell progenitor fate. This study characterized the proteome of epithelial and endothelial cells from the BPD lung for the first time, identifying population-specific changes in BPD pathogenesis.

BPD↗