Human testicular peritubular cells (HTPCs) are smooth muscle cells, which in the testis form a small compartment surrounding the seminiferous tubules. Contractions of HTPCs are responsible for sperm transport, HTPCs contribute to spermatogenesis, have immunological roles and are a site of glucocorticoid receptor expression. Importantly, HTPCs maintain their characteristics in vitro, and thus can serve as an experimental window into the male gonad. Previously we reported consequences of 3-day treatment with Dexamethasone (Dex), a synthetic glucocorticoid and multi-purpose anti-inflammatory drug. However, as glucocorticoid therapies in man often last longer, we now studied consequences of a prolonged 7-day exposure to 1 µM Dex. Combining live cell imaging with quantative proteomics of samples taken from men, we confirmed our recent findings but more importantly, found numerous novel proteomic alterations induced by prolonged Dex treatment. The comparison of the 7-day treatment with the 3-day treatment dataset revealed that extracellular matrix- and focal adhesion-related proteins become more prominent after 7 days of treatment. In contrast, extended stimulation is, for example, associated with a decrease of proteins related to cholesterol and steroid metabolism. Our dataset, which describes phenotypic and proteomic alterations, is a valuable resource for further research projects investigating effects of Dex on human testicular cells.

MALDI mass spectrometry imaging (MALDI imaging) uniquely advances cancer research, by measuring spatial distribution of endogenous and exogenous molecules directly from tissue sections. These molecular maps provide valuable insights into basic and translational cancer research, including tumor biology, tumor microenvironment, biomarker identification, drug treatment, and patient stratification. Despite its advantages, MALDI imaging is underutilized in studying rare cancers. Sarcomas, a group of malignant mesenchymal tumors, pose unique challenges in medical research due to their complex heterogeneity and low incidence, resulting in understudied subtypes with suboptimal management and outcomes. In this review, we explore the applicability of MALDI imaging in sarcoma research, showcasing its value in understanding this highly heterogeneous and challenging rare cancer. We summarize all MALDI imaging studies in sarcoma to date, highlight their impact on key research fields, including molecular signatures, cancer heterogeneity, and drug studies. We address specific challenges encountered when employing MALDI imaging for sarcomas, and propose solutions, such as using formalin-fixed paraffin-embedded tissues, and multiplexed experiments, and considerations for multi-site studies and digital data sharing practices. Through this review, we aim to spark collaboration between MALDI imaging researchers and clinical colleagues, to deploy the unique capabilities of MALDI imaging in the context of sarcoma.

This Dataset Brief describes the computational prediction of protein structures for the ctenophore Mnemiopsis leidyi. Here, we report the proteome-scale generation of 15,333 protein structure predictions using AlphaFold, as well as an updated implementation of publicly available search, manipulation, and visualization tools for these protein structure predictions through the Mnemiopsis Genome Project Portal (https://research.nhgri.nih.gov/mnemiopsis). The utility of these predictions is demonstrated by highlighting comparisons to experimentally determined structures for the light-sensitive protein mnemiopsin 1 and the ionotropic glutamate receptor (iGluR). The application of these novel protein structure prediction methods will serve to further position non-bilaterian species such as Mnemiopsis as powerful model systems for the study of early animal evolution and human health.

Small proteins (SPs) are a unique group of proteins that play crucial roles in many important biological processes. Exploring the biological function of SPs is necessary. In this study, the InterPro tool and the maximum correlation method were utilized to analyze functional domains of SPs. The purpose was to identify important functional domains that can indicate the essential differences between small and large protein sequences. First, the small and large proteins were represented by their functional domains via a one-hot scheme. Then, the MaxRel method was adopted to evaluate the relationships between each domain and the target variable, indicating small or large protein. The top 36 domain features were selected for further investigation. Among them, 14 were deemed to be highly related to SPs because they were annotated to SPs more frequently than large proteins. We found the involvement of functional domains, such as ubiquitin-conjugating enzyme/RWD-like, nuclear transport factor 2 domain, and alpha subunit of guanine nucleotide-binding protein (G-protein) in regulating the biological function of SPs. The involvement of these domains has been confirmed by other recent studies. Our findings indicate that protein functional domains may regulate small protein-related functions and predict their biological activity.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophectodermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF). We show these loaded NVs elicit EGFR-mediated effects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF-mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrates feasibility in enhancing the functional potency of NVs in the context of embryo implantation.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

The alpha7 nicotinic acetylcholine receptor (α7 nAChR; CHRNA7) is expressed in the nervous system and in non-neuronal tissues. Within the central nervous system, it is involved in various cognitive and sensory processes such as learning, attention, and memory. It is also expressed in the cerebellum, where its roles are; however, not as well understood as in the other brain regions. To investigate the consequences of absence of CHRNA7 on the cerebellum proteome, we performed a quantitative nano-LC-MS/MS analysis of samples from CHRNA7 knockout (KO) mice and corresponding wild type (WT) controls. Liver, an organ which does not express this receptor, was analyzed, in comparison. While the liver proteome remained relatively unaltered (three proteins more abundant in KOs), 90 more and 20 less abundant proteins were detected in the cerebellum proteome of the KO mice. The gene ontology analysis of the differentially abundant proteins indicates that the absence of CHRNA7 leads to alterations in the glutamatergic system and myelin sheath in the cerebellum. In conclusion, our dataset provides new insights in the role of CHRNA7 in the cerebellum, which may serve as a basis for future in depth-investigations.

Glycosylation, the major post-translational modification of proteins, significantly increases the diversity of proteoforms. Glycans are involved in a variety of pivotal structural and functional roles of proteins, and changes in glycosylation are profoundly connected to the progression of numerous diseases. Mass spectrometry (MS) has emerged as the gold standard for glycan and glycopeptide analysis because of its high sensitivity and the wealth of fragmentation information that can be obtained. Various separation techniques have been employed to resolve glycan and glycopeptide isomers at the front end of the MS. However, differentiating structures of isobaric and isomeric glycopeptides constitutes a challenge in MS-based characterization. Many reports described the use of various ion mobility-mass spectrometry (IM-MS) techniques for glycomic analyses. Nevertheless, very few studies have focused on N- and O-linked site-specific glycopeptidomic analysis. Unlike glycomics, glycoproteomics presents a multitude of inherent challenges in microheterogeneity, which are further exacerbated by the lack of dedicated bioinformatics tools. In this review, we cover recent advances made towards the growing field of site-specific glycosylation analysis using IM-MS with a specific emphasis on the MS techniques and capabilities in resolving isomeric peptidoglycan structures. Furthermore, we discuss commonly used software that supports IM-MS data analysis of glycopeptides.

Understanding the molecular changes associated with the aged brain forms the basis for developing potential strategies for slowing cognitive decline associated with normal aging. Focusing on the hippocampus, a critical brain region involved in learning and memory, we employed tandem mass tag methodology to investigate global proteomic changes that occur in advanced-aged (20-month) versus young (3-month) C57BL/6 male mice. Our analysis revealed the upregulation of 236 proteins in the old hippocampal proteome, including those enriched within several age-related processes, such as the adaptive immune response and molecular metabolic pathways, whereas downregulated proteins (88 in total) are mainly involved in axonogenesis and growth cone-related processes. Categorizing proteins by cell-type enrichment in the brain identified a general upregulation of proteins preferentially expressed in microglia, astrocytes, and oligodendrocytes. In contrast, proteins with neuron-specific expression displayed an overall age-related downregulation. By integrating our proteomic with our previously published transcriptomic data, we discovered a mild but significant positive correlation between mRNA and protein expression changes in the aged hippocampus. Therefore, this proteomic data is a valuable additional resource for further understanding age-related molecular mechanisms.