James M. Fulcher

The extracellular matrix (ECM) serves critical structural and functional purposes within tissues, but its spatial arrangement and composition across functional tissue units (FTUs) have not been fully elucidated. Here, we present a streamlined workflow for spatial matrisomics that enables confident identification of ECM peptides directly from tissue sections. By processing adjacent formalin-fixed, paraffin-embedded (FFPE) tissue sections in parallel, we generate MALDI-MSI-ready samples and matched LC-MS/MS data sets using a unified collagenase-based digestion strategy. This parallelized approach reduces sample handling and supports construction of a robust ECM peptide reference library. We further introduce an automated, open-source mass-matching pipeline that integrates LC-MS/MS peptide identifications with MALDI-MSI features to facilitate high-confidence annotation. Applying this workflow to human kidney tissues and pairing MSI data with AI-driven digital pathology, we resolve FTU-specific ECM profiles, including glomerular enrichment of Tenascin-C and Albumin, and tubular enrichment of COL1A2 and COL3A1 peptides. Across samples, we observe both conserved and variable ECM patterns, emphasizing the importance of spatially resolved analyses for understanding tissue ECM heterogeneity. Altogether, this approach advances spatial matrisomics by increasing throughput, reducing manual data curation, and enhancing confidence in ECM peptide identifications, which can enable future applications in studying fibrosis and kidney disease, for example.

With a sub-3-µm cell and a 27-Mbp diploid genome, the small but mighty Picochlorum celeri doubles within three hours and exceeds 30 g m ⁻² d ⁻¹ outdoors with CO ₂ supplied. In climate-simulating photobioreactors, productivity declined from 39 g m ⁻² d ⁻¹ under 2.25% CO ₂ to near zero in air and resumed within hours of CO ₂ restoration with photosystem II photochemistry largely retained. We profiled the transcriptome, proteome, phosphoproteome, ubiquitinome, acetylome, metabolome and lipidome, including the first site-resolved green-algal ubiquitinome. In air, the Rubisco large subunit tripled to 7.4% of protein while its two small-subunit isoforms, 70% identical, exchanged near-reciprocally, a novel CO ₂ -dependent switch that we propose retunes Rubisco to low CO ₂ . Photorespiratory enzymes increased in abundance whereas proteins involved in external nitrogen assimilation decreased, consistent with increased reliance on internal nitrogen recycling as carbon fixation became limiting. Transcript and protein responses were nearly uncoupled, with cytosolic ribosomal proteins declining despite increased transcripts and plastid ribosomal proteins spared. Phosphorylation was extensively remodeled, as were the lysine modifications ubiquitination, acetylation and CO ₂ -dependent carbamylation, where a CO ₂ -competition assay identified 86 bicarbonate-protected lysines, including RbcL K252. CO ₂ limitation therefore imposes a reversible growth arrest that reduces cytosolic translational investment while preserving photosynthetic capacity for rapid recovery.

Hydrogenosomes are mitochondrion-derived organelles that produce ATP and H ₂ to support energy metabolism in anaerobic eukaryotes. H ₂ production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H ₂ production in anaerobic eukaryotes remain incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) ⁺ and link electron transfer to H ₂ formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H ₂ production in the AF Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H ₂ :NAD ⁺ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H ₂ formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE and NuoF) in C. churrovis and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H ₂ directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme known from anaerobic prokaryotes. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex for H ₂ production in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H ₂ production and altering fungal metabolism. IMPORTANCE: H ₂ production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H ₂ production in Neocallimastix was first reported, the molecular mechanism for hydrogenosomal H ₂ production and redox balance remains unclear. We demonstrate that AF produce H ₂ from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H ₂ production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H ₂ -producing systems.

The plant root tip is a highly specialized region where cells transition from stem cells to differentiated types and respond to environmental cues. Yet, cell-type-specific proteomic responses to abiotic stress in field-grown plants remain poorly understood. This study generated proteomes for five root-tip cell types in switchgrass (Panicum virgatum), including (1) quiescent center and stem cell niche (Quc), (2) protodermal epidermal cells in the meristematic zone (PEC), (3) epidermal cells in the transition and elongation zones (Epi), (4) peripheral root cap cells (PRC) and (5) columella root cap cells (Col), and characterized their drought-responsive profiles. Root tips from seven-year-old field-grown “Alamo” plants under well-watered or long-term drought conditions were analyzed using laser capture microdissection (LCM) coupled with nanoPOTS and nano-LC-MS. In well-watered samples, 6336–6640 proteins were quantified per cell type, and principal component analysis confirmed distinct proteomic identities. Drought-stressed samples, collected from fewer cells per type, yielded 1109–3298 proteins, with 4,493 proteins quantified overall and 935 shared across all cell types. After median normalization of peptide abundance, differentially abundant proteins (DAPs) were identified using Welch’s t test (|log2FC| > 1, adjusted p ≤ 0.05), revealing cell-type-specific drought responses, with the largest numbers of DAPs in Quc and Col cells. DAPs were associated with phytohormone biosynthesis and signaling (auxin, cytokinin, jasmonic acid), DNA repair and mitosis (notably enriched in Quc), and metabolic pathways involving amino acids, carbohydrates, and lipids. Stress-responsive proteins generally increased, whereas proteins linked to translation decreased across all cell types. Overall, this work provides the first spatially resolved, cell-type-specific root-tip proteomes from field-grown switchgrass, offering new insight into distinct cellular strategies underlying drought tolerance.

Genome-scale metabolic models (GEMs) are powerful tools for predicting cellular phenotypes and guiding microbial strain engineering, yet broad adoption remains challenging due to the computational expertise required. To overcome that, we present ChatGEM, an agentic platform that enables interactive GEM simulation through natural language. Built on the multi-agent ADEPT framework, ChatGEM integrates COBRApy within a retrieval-augmented generation (RAG) architecture that coordinates code generation and execution through specialized agents. Benchmarking across three tasks of increasing complexity showed that RAG-enabled code generation improved the mean overall performance score from 2.63 to 4.20 while reducing the execution time significantly starting from routine to complex tasks. Application of ChatGEM using an enzyme-constrained GEM (ecGEM) for four engineered Pseudomonas putida KT2440 strains identified the constitutive strain as the optimal chassis for succinate overproduction using a succinate leakage index - a prediction observed experimentally. Therefore, ChatGEM democratizes metabolic modeling by enabling researchers without computational expertise to perform sophisticated GEM-based analyses through natural language, and, hence, accelerating scientific discovery.

Hydrogenosomes are mitochondria-derived organelles that produce ATP and H ₂ to support energy metabolism in anaerobic eukaryotes. H ₂ production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H ₂ production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) ⁺ and link electron transfer to H ₂ formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H ₂ production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H ₂ :NAD ⁺ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H ₂ formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H ₂ directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H ₂ production and altering fungal metabolism. IMPORTANCE: H ₂ production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H ₂ production from AF was first reported, the molecular mechanism for hydrogenosomal H ₂ production and redox balance remains unclear. We demonstrate that AF produce H ₂ from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H ₂ production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H ₂ -producing systems.

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

Mass spectral libraries have become essential resources for training deep learning (DL) models for spectral prediction and de novo sequencing in bottom-up mass spectrometry (BU-MS). Compared with BU-MS, top-down MS (TD-MS) offers unique advantages for characterizing intact proteoforms by analyzing proteoforms without enzymatic digestion. Despite these advantages, large-scale spectral libraries for TD-MS are currently lacking. Here we present TopRepo, the first comprehensive and publicly available repository of TD-MS spectra, comprising more than 12 million spectra acquired from 12 species across seven types of mass spectrometers. Using TopRepo, we constructed a large-scale top-down spectral library containing over 5 million spectra with proteoform and fragment-ion annotations. We demonstrate that TopRepo enables pan-dataset analyses of N-terminal processing, mass shifts, and other proteoform characteristics identified by TD-MS. Furthermore, we show that the TopRepo spectral library substantially improves proteoform identification through spectral library searching and supports the training of DL models for high-accuracy top-down spectral prediction.

Laser capture microdissection (LCM) - based spatial mass spectrometry proteomics is a rapidly emerging technique with strong potential for use in formalin-fixed, paraffin-embedded (FFPE) tissues. Several sample-preparation methods have been developed to decrosslink FFPE proteins for spatial proteomics; however, residual crosslinks often remain, and depth can remain impaired relative to fresh frozen tissue samples. To increase proteome coverage in spatially resolved LCM-FFPE samples, we investigated a panel of chemical compounds with the potential to catalyze the decrosslinking of nucleophilic functional groups on proteins. Systematic screening and optimization of temperature, incubation time, and reagent concentration led to the identification of 3,4-diaminobenzoic acid as an effective agent for improving proteome coverage in FFPE pancreatic tissue. This compound could boost precursor identifications by more than 10% at both reduced (70 °C) and high (90 °C) temperatures. Application of this chemical-decrosslinking strategy to a pancreatic ductal adenocarcinoma tissue section enabled the identification of numerous cell-type–enriched proteins with clinical and therapeutic relevance. Taken together, our findings show that chemical decrosslinking can increase proteome coverage in FFPE tissues, thereby advancing our understanding of tissue microenvironments in physiological and pathological contexts.