Accurate, in-depth mapping of proteins on whole-tissue levels provides comprehensive insights into the spatially-organized regulatory processes/networks in tissues, but is challenging. Here we describe a micro-scaffold assisted spatial proteomics (MASP) strategy, based on spatially-resolved micro-compartmentalization of tissue using a 3D-printed micro-scaffold, capable of mapping thousands of proteins across a whole-tissue slice with excellent quantitative accuracy/precision. The pipeline includes robust tissue micro-compartmentalization with precisely-preserved spatial information, reproducible procurement and preparation of the micro-specimens, followed by sensitive LC-MS analysis and map generation by a MAsP app. The mapping accuracy was validated by comparing the MASP-generated maps of spiked-in peptides and brain-region-specific markers with known patterns, and by correlating the maps of the two protein components of the same heterodimer. The MASP was applied in mapping >5000 cerebral proteins in the mouse brain, encompassing numerous important brain markers, regulators, and transporters, where many of these proteins had not previously been mapped on the whole-tissue level.

BACKGROUND: ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, 13), primarily synthesized in hepatic stellate and endothelial cells, plays a pivotal role in regulation of hemostasis by proteolytic cleavage of von Willebrand factor. Severe deficiency of plasma ADAMTS13 activity may result in thrombotic thrombocytopenic purpura, a potentially fatal blood disorder. ADAMTS13 undergoes posttranslational modifications including glycosylation, citrullination, oxidation. The present study determines the impact of arginine methylation by PRMT1 (protein arginine methyltransferase 1) on ADAMTS13 secretion and function. METHODS: Cell culture, recombinant protein, biochemical analysis, site-directed mutagenesis, and animal models were utilized. RESULTS: An inhibition of arginine methylation by a type I methyl transferase PRMT inhibitor (MS023) in HEK (human embryonic kidney) 293 cells expressing recombinant ADAMTS13 and in mice results in a significant reduction of ADAMTS13 secretion, but the secreted ADAMTS13 shows an increased specific activity; conversely, an overexpression of PRMT1 in HEK-293 cells and in transgenic mice results in an increase of ADAMTS13 secretion, but the secreted ADAMTS13 exhibits a significantly reduced specific activity. The altered ADAMTS13 activity appeared to be related to its conformational changes. LC-MS/MS (liquid chromatography with tandem mass spectrometry) identified greater than100 arginine methylation events on purified recombinant ADAMTS13. Site-directed mutagenesis performed on 5 highly conserved methylation sites (R193, R498, R692, R1123, and R1206) identifies the critical role of R1206 in ADAMTS13 function. The ADAMTS13 R1206K variant exhibits a 4- to 5-fold increase of specific activity, likely resulting from an alleviation of allosteric inhibition. CONCLUSIONS: These results demonstrate the crucial role of arginine methylation in ADAMTS13 secretion and function. Our findings may shed new light on the mechanism of allosteric regulation of ADAMTS13, which may have a therapeutic implication.

Although traumatic brain injury (TBI) causes hospitalizations and mortality worldwide, there are no approved neuroprotective treatments, partly due to a poor understanding of the molecular mechanisms underlying TBI neuropathology and neuroprotection. We previously reported that the administration of low-dose methamphetamine (MA) induced significant functional/cognitive improvements following severe TBI in rats. We further demonstrated that MA mediates neuroprotection in part, via dopamine-dependent activation of the PI3K-AKT pathway. Here, we further investigated the proteomic changes within the rat cortex and hippocampus following mild TBI (TM), severe TBI (TS), or severe TBI plus MA treatment (TSm) compared to sham operated controls. We identified 402 and 801 altered proteins (APs) with high confidence in cortical and hippocampal tissues, respectively. The overall profile of APs observed in TSm rats more closely resembled those seen in TM rather than TS rats. Pathway analysis suggested beneficial roles for acute signaling through IL-6, TGFβ, and IL-1β. Moreover, changes in fibrinogen levels observed in TSm rats suggested a potential role for these proteins in reducing/preventing TBI-induced coagulopathies. These data facilitate further investigations to identify specific pathways and proteins that may serve as key targets for the development of neuroprotective therapies.

Reprogramming of the androgen receptor (AR) cistrome is associated with prostate cancer progression, and advanced castration-resistant prostate cancers (CRPC) tend to rely on reprogrammed/noncanonical AR signaling that remains active under treatment with AR signaling inhibitors (ARSI). In this study, we identified ecotropic viral integration site 1 (EVI1), an oncogenic nuclear transcription factor (TF) encoded by MECOM, as an AR-recruited coactivator of noncanonical signaling. In prostate cancer, MECOM was exclusively overexpressed in both CRPC and enzalutamide-resistant CRPC and interacted with AR in the nucleus. MECOM depletion in prostate cancer cells decreased proliferation, altered cell survival transcriptional programs, and reduced the number of super-enhancers (SE), leading to a dynamic change in the SE landscape and a decrease in the expression of SE-regulated oncogenic TFs, along with increased proapoptotic signatures. Notably, cells overexpressing MECOM and its protein, EVI1, were susceptible to PARP inhibitors (PARPi) regardless of their DNA damage response or homologous recombination repair (HRR) gene mutation status. These insights reveal the crucial role of EVI1 in regulating cell survival within the context of an AR-reprogrammed chromatin landscape. More importantly, the findings suggest that MECOM overexpression may be another biomarker that could significantly broaden the use of PARPis beyond those with HRR gene mutations. Significance: MECOM is a driver of androgen receptor inhibitor resistance in castrate-resistant prostate cancer that promotes susceptibility to PARP inhibition, positioning MECOM as a biomarker in patients without homologous recombination repair gene mutations.

Cardiovascular function is tightly linked to tissue architectures, where the spatial organization of cells, extracellular matrix (ECM), vascular networks, and remodeling processes governs physiological performance and disease progression. Spatial proteomics has, therefore, emerged as a powerful framework for understanding cardiovascular biology and cardiovascular disease mechanisms by revealing spatially organized protein regulation across various physiological and pathological states. In this review, we focus on spatial proteomics strategies most relevant to cardiovascular research and discuss their applications through representative examples. These approaches can be broadly categorized into region-of-interest–based methods, which enable precise characterization of localized cellular heterogeneity, and tissue mapping strategies, which capture spatial organization and biologically relevant region-to-region variability across larger tissue domains. In addition, spatial proteomics platforms differ in their capacity for targeted or untargeted protein analysis, influencing both proteome coverage and their suitability for hypothesis-driven versus discovery-based studies. We evaluate the strengths and limitations of state-of-the-art technologies across 3 key parameters, molecular depth, spatial coverage, and spatial resolution, and discuss how these parameters shape study design and biological understanding. Building on these considerations, we argue that a comprehensive understanding of cardiovascular tissue biology requires spatial proteomics strategies that capture both localized molecular details and spatial organization across large tissue areas, as neither alone is sufficient to explain complex tissue behavior. We propose an integrated workflow in which untargeted, whole-tissue mapping of thousands of proteins is first used to unbiasedly discover spatial patterns and generate hypotheses by identifying candidate regions and proteins of interest, followed by hypothesis testing and validation using high-precision region-of-interest–based proteomics and targeted protein imaging. This sequential framework leverages the complementary strengths of tissue-wide mapping and region-of-interest–based approaches to provide multiscale, mechanistic insights into spatially organized disease processes. Finally, we discuss emerging directions that are poised to expand the scope of spatial proteomics in cardiovascular research.