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Extracellular vesicles (EVs)-mediated cellular communication plays a role in cancer development and progression. This study focuses on identifying glioblastoma-specific EV protein markers through a comparative mass spectrometry bottom-up proteomic analysis of the LN-229 cell line and human neurons, astrocytes, and endothelial brain cells (HEBCs) using timsTOF Pro 2 instrument. The statistically significant upregulated proteins with fold change greater than 2 in the glioblastoma-derived EVs were clustered based on physical and functional interactions using the STRING database and analyzed using Gene Ontology enrichment. LN229-derived EVs contained an average of 2,635 proteins, while human astrocytes, neurons, and HEBC encapsulated 2,647, 716, and 2285 proteins, respectively. NanoParticle Tracking Analysis indicated that glioblastoma-derived EVs exhibited greater size variability compared to EVs from healthy cells. Statistical analysis identified 25 statistically significant proteins with increased levels in LN229 EVs relative to at least two healthy cell lines suggesting their potential as glioblastoma markers. Functional clustering using the STRING database and GO analysis indicated involvement in epigenetic regulation, metastasis, angiogenesis, and protein folding. Post-translational modification analysis identified a subset of 17 proteins unique to the cancer-derived EVs involved in chromatin regulation, extracellular matrix remodeling, and basement membrane organization pathways, highlighting their role in tumor progression. 

Abstract Alkyne tags have been widely used for the enrichment of labeled proteins to enable their profiling at a proteome‐wide scale by mass spectrometry. The key component in the enrichment process is an azido‐terminated cleavable linker for capturing the labeled proteins/peptides via click reaction. Herein, we report a new efficient click linker (APAbiotin) featuring an acid‐cleavable acetal linkage end‐caped with a highly reactive picolyl azido head and a biotin handle for anchoring onto streptavidin‐coated supports. Using an amine‐reactive probe to profile the proteome structural changes in livingS. cerevisiaecells within 5 minutes of heat shock, we demonstrated that the linker allowed identification of >9400 labeled sites, among which 457–1656 with significantly altered reactivity upon heat shock. This study represented the first chemical labeling mass spectrometry (CL–MS)‐based profiling of proteome structural changes in living cells in response to external stimuli. Data are available via ProteomeXchange with identifier PXD051279. 

Abstract Rabbits have been widely used for studying ocular physiology and pathology due to their relatively large eye size and similar structures with human eyes. Various rabbit ocular disease models, such as dry eye, age-related macular degeneration, and glaucoma, have been established. Despite the growing application of proteomics in vision research using rabbit ocular models, there is no spectral assay library for rabbit eye proteome publicly available. Here, we generated spectral assay libraries for rabbit eye compartments, including conjunctiva, cornea, iris, retina, sclera, vitreous humor, and tears using fractionated samples and ion mobility separation enabling deep proteome coverage. The rabbit eye spectral assay library includes 9,830 protein groups and 113,593 peptides. We present the data as a freely available community resource for proteomic studies in the vision field. Instrument data and spectral libraries are available via ProteomeXchange with identifier PXD031194.