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Growing evidence supports the importance of extracellular vesicle (EV) as mediators of communication in pathological processes, including those underlying respiratory disease. However, establishing methods for isolating and characterizing EVs remains challenging, particularly for respiratory samples. This study set out to address this challenge by comparing different EV isolation methods and evaluating their impacts on EV yield, markers of purity, and proteomic signatures, utilizing equine/horse bronchoalveolar lavage samples. Horses can serve as effective translational animal models for respiratory studies due to similarities with human immune responses, shared environmental exposures, and naturally occurring respiratory diseases including asthma. Further, horses are long-lived large animals that allow for longitudinal sample collection, and provide large sample volume and cell yield, which are particularly useful since EV research is commonly limited by low sample yields. Here, EVs were isolated from horse bronchoalveolar lavage fluid (BALF) using four different methods (ultracentrifugation, microcentrifugation, and two sizes of size exclusion chromatography columns) and characterized by measuring particle counts, EV purity, total protein yield, and proteomic cargo, with a specific focus on vesicle surface marker expression potentially informing cell type of origin. We found that size exclusion chromatography yielded the highest particle counts, greatest EV purity markers and elevated vesicle surface marker expression. Overall proteomic profiles differed across isolation methods, with size exclusion chromatography clustering separately from centrifugation. Taken together, our results demonstrate that different isolation methods impact characteristics of EVs, notably that size exclusion chromatography, compared to centrifugation methods, resulted in higher EV purity and better characterized proteomic diversity, including information on EV cell-of-origin. This is the first study to characterize proteomic profiles of EVs following different isolation methods using equine BALF. The results of this study will pave the way for future studies using equine and human samples to characterize respiratory tract EVs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Hickman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Wildfires adversely impact air quality and public health worldwide. Exposures to wildfire smoke are linked to adverse health outcomes, including cardiopulmonary diseases. Critical research gaps remain surrounding the underlying biological pathways leading to wildfire-induced health effects. The regulation of intercellular communication and downstream toxicity driven by extracellular vesicles (EVs) is an important, understudied biological mechanism. This study investigated EVs following a wildfire smoke-relevant in vitro exposure. We hypothesized that woodsmoke (WS) would alter the proteomic content of EVs secreted in organotypic in vitro airway models. Exposures were carried out using a tri-culture model of alveolar epithelial cells, fibroblasts, and endothelial cells and a simplified co-culture model of alveolar epithelial cells and fibroblasts to inform responses across different cell populations. Epithelial cells were exposed to WS condensate and EVs were isolated from basolateral conditioned medium following 24 h exposure. WS exposure did not influence EV particle characteristics, and it moderately increased EV count. Exposure caused the differential loading of 25 and 35 proteins within EVs collected from the tri- and co-culture model, respectively. EV proteins involved in extracellular matrix degradation and wound healing were consistently modulated across both models. However, distinct proteins involved in the wound healing pathway were altered between models, suggesting unique but concerted efforts across cell types to communicate in response to injury. These findings demonstrate that a wildfire-relevant exposure alters the EV proteome and suggest an impact on EV-mediated intercellular communication. Overall, results demonstrate the viability of organotypic approaches in evaluating EVs to investigate exposure-induced biomarkers and underlying mechanisms. Findings also highlight the impact of differences in the biological complexity of in vitro models used to evaluate the effects of inhaled toxicants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF) βTrCP . However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCF Cyclin F , demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/βTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the advantages of fewer missing values by collecting fragment ion data on all analytes in the sample as well as the potential for deeper coverage, the adoption of data-independent acquisition (DIA) in proteomics core facility settings has been slow. The Association of Biomolecular Resource Facilities conducted a large interlaboratory study to evaluate DIA performance in proteomics laboratories with various instrumentation. Participants were supplied with generic methods and a uniform set of test samples. The resulting 49 DIA datasets act as benchmarks and have utility in education and tool development. The sample set consisted of a tryptic HeLa digest spiked with high or low levels of 4 exogenous proteins. Data are available in MassIVE MSV000086479. Additionally, we demonstrate how the data can be analyzed by focusing on 2 datasets using different library approaches and show the utility of select summary statistics. These data can be used by DIA newcomers, software developers, or DIA experts evaluating performance with different platforms, acquisition settings, and skill levels., Competing Interests: Conflict of Interest Disclosures: BCS is a founder and shareholder in Proteome Software, which operates in the field of proteomics. BS is the CEO at Bioinformatics Solutions Inc., which creates software for the field of proteomics. The other authors declare no competing interests., (Copyright © 2023 Association of Biomolecular Resource Facilities. All rights reserved.)

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis., (© 2022 Federation of American Societies for Experimental Biology.)

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)