Mass Spectrometry-based Proteomic Analysis Research Articles

HMGB3 and SUB1 Bind to and Facilitate the Repair of N2-Alkylguanine Lesions in DNA.

N2-Alkyl-2'-deoxyguanosine (N2-alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N2-alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N2-alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate N2-alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to N2-nBu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N2-BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans-N2-BPDE-dG; the protein, however, does not distinguish cis-N2-BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans-N2-BPDE-dG, but not its cis counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove N2-alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N2-BPDE-dG.

Characteristics and Regulation of Human Eosinophil ETosis In Vitro.

Cytolytic ETosis is a type of programmed cell death distinct from apoptosis and necrosis and plays a major role in the innate immune system and disease progression. Through the process of ETosis, cells release their chromatin with diverse antimicrobial proteins into the extracellular milieu, forming extracellular traps (ETs). Although ETosis has been reported in several leukocyte types, few studies have compared ETosis and the component proteins of ETs in leukocytes. The aim of this study was to better understand the characteristics of eosinophil ETosis (EETosis) compared with other leukocytes. We isolated human blood eosinophils, neutrophils, basophils, monocytes, and lymphocytes and stimulated them with known ETosis inducers, a protein kinase C activator PMA, or a calcium ionophore A23187. Both stimuli induced eosinophil cell death and ET release after 180 minutes of stimulation in a NADPH-oxidase-dependent manner. PMA also induced NADPH-oxidase-dependent ETosis in neutrophils, whereas little or no significant ETosis was observed in basophils, monocytes, or lymphocytes at 180 minutes. Mass spectrometry-based proteomic analysis of eosinophil- and neutrophil-derived ETs identified 997 and 1415 proteins, respectively. Among the physiological stimuli tested, immobilized IgA and IgG induced EETosis. C-C motif chemokine ligand 11 (CCL11) and interleukin 5 (IL-5) were weak inducers of EETosis, but co-stimulation significantly induced rapid EETosis. Under high serum or albumin conditions, co-stimulation with CCL11 and IL-5 paradoxically prolonged cell survival by preventing spontaneous apoptosis. This study provides an in-depth characterization of EETosis and highlights the precise regulation of eosinophil survival and cell death pathways.

Proteomic profile and predictive markers of outcome in patients with subarachnoid hemorrhage

BackgroundThe molecular mechanisms underlying development of posthemorrhagic hydrocephalus (PHH) following subarachnoid hemorrhage (SAH) remain incompletely understood. Consequently, treatment strategies tailored towards the individual patient remain limited. This study aimed to identify proteomic cerebrospinal fluid (CSF) biomarkers capable of predicting shunt dependency and functional outcome in patients with SAH in order to improve informed clinical decision making.MethodsVentricular CSF samples were collected twice from 23 patients with SAH who required external ventricular drain (EVD) insertion (12 patients with successful EVD weaning, 11 patients in need of permanent CSF shunting due to development of PHH). The paired CSF samples were collected acutely after ictus and later upon EVD removal. Cisternal CSF samples were collected from 10 healthy control subjects undergoing vascular clipping of an unruptured aneurysm. All CSF samples were subjected to mass spectrometry-based proteomics analysis. Proteomic biomarkers were quantified using area under the curve (AUC) estimates from a receiver operating curve (ROC).ResultsCSF from patients with SAH displayed a distinct proteomic profile in comparison to that of healthy control subjects. The CSF collected acutely after ictus from patients with SAH was moreover distinct from that collected weeks later but appeared similar in the weaned and shunted patient groups. Sixteen unique proteins were identified as potential predictors of shunt dependency, while three proteins were identified as potential predictors of functional outcome assessed six months after ictus with the modified Rankin Scale.ConclusionsWe here identified several potential proteomic biomarkers in CSF from patients with SAH capable of predicting (i) shunt dependency and thus development of PHH and (ii) the functional outcome assessed six months after ictus. These proteomic biomarkers may have the potential to aid clinical decision making by predicting shunt dependency and functional outcome following SAH.

SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

Ewing’s sarcoma (ES) represents a rare yet exceedingly aggressive neoplasm that poses a significant health risk to the pediatric and adolescent population. The clinical outcomes for individuals with relapsed or refractory ES are notably adverse, primarily attributed to the constrained therapeutic alternatives available. Despite significant advancements in the field, molecular pathology-driven therapeutic strategies have yet to achieve a definitive reduction in the mortality rates associated with ES. Consequently, there exists an imperative need to discover innovative therapeutic targets to effectively combat ES. To reveal the mechanism of the SETD8 (also known as lysine methyltransferase 5A) inhibitor UNC0379, cell death manners were analyzed with different inhibitors. The contributions of SETD8 to the processes of apoptosis and ferroptosis in ES cells were evaluated employing the histone methyltransferase inhibitor UNC0379 in conjunction with RNA interference techniques. The molecular regulatory mechanisms of SETD8 in ES were examined through the application of RNA sequencing (RNA-seq) and mass spectrometry-based proteomic analysis. Moreover, nude mouse xenograft models were established to explore the role of SETD8 in ES in vivo. SETD8, a sole nucleosome-specific methyltransferase that catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), was found to be upregulated in ES, and its overexpression was associated with dismal outcomes of patients. SETD8 knockdown dramatically induced the apoptosis and ferroptosis of ES cells in vitro and suppressed tumorigenesis in vivo. Mechanistic investigations revealed that SETD8 facilitated the nuclear translocation of YBX1 through post-transcriptional regulatory mechanisms, which subsequently culminated in the transcriptional upregulation of RAC3. In summary, SETD8 inhibits the apoptosis and ferroptosis of ES cells through the YBX1/RAC3 axis, which provides new insights into the mechanism of tumorigenesis of ES. SETD8 may be a potential target for clinical intervention in ES patients.

Extracellular C1qbp inhibits myogenesis by suppressing NFATc1

Aging and lack of exercise are the most important etiological factors for muscle loss. We hypothesized that new factors that contribute to muscle loss could be identified from ones commonly altered in expression in aged and exercise-limited skeletal muscles. Mouse gastrocnemius muscles were subjected to mass spectrometry-based proteomic analysis. The muscle proteomes of hindlimb-unloaded and aged mice were compared to those of exercised and young mice, respectively. C1qbp expression was significantly upregulated in the muscles of both hindlimb-unloaded and aged mice. In vitro myogenic differentiation was not affected by altering intracellular C1qbp expression but was significantly suppressed upon recombinant C1qbp treatment. Additionally, recombinant C1qbp repressed the protein level but not the mRNA level of NFATc1. NFATc1 recruited the transcriptional coactivator p300, leading to the upregulation of acetylated histone H3 levels. Furthermore, NFATc1 silencing inhibited p300 recruitment, downregulated acetylated histone H3 levels, and consequently suppressed myogenic differentiation. The expression of C1qbp was inversely correlated with that of NFATc1 in the gastrocnemius muscles of exercised or hindlimb-unloaded, and young or aged mice. These findings demonstrate a novel role of extracellular C1qbp in suppressing myogenesis by inhibiting the NFATc1/p300 complex. Thus, C1qbp can serve as a novel therapeutic target for muscle loss.

A hot and cold tumor‑related prognostic signature for stage II colorectal cancer.

Globally, colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. Based on the presence of immune cell infiltration in the tumor microenvironment, CRC can be divided into immunologically 'hot' or 'cold' tumors, which in turn leads to the differential efficacy of immunotherapy. However, the immune characteristics of hot and cold CRC tumors remain largely elusive, prompting further investigation of their properties regarding the tumor microenvironment. In the present study, a predictive model was developed based on the differential expression of proteins between cold and hot CRC tumors. First, the differentially expressed proteins (DEPs) were identified using digital spatial profiling and mass spectrometry-based proteomics analysis, and the pathway features of the DEPs were analyzed using functional enrichment analysis. A novel eight-gene signature prognostic risk model was developed (IDO1, MAT1A, NPEPL1, NT5C, PTGR2, RPL29, TMEM126A and TUBB4B), which was validated using data obtained from The Cancer Genome Atlas. The results revealed that the risk score of the eight-gene signature acted as an independent prognostic indicator in patients with stage II CRC (T3-4N0M0). It was also found that a high-risk score in the eight-gene signature was associated with high immune cell infiltration in patients with CRC. Taken together, these findings revealed some of the differential immune characteristics of hot and cold CRC tumors, and an eight-gene signature prognostic risk model was developed, which may serve as an independent prognostic indicator for patients with stage II CRC (T3-4N0M0).

An optimized protocol for pig intestinal mucosa proteomics

The overall well-being of organisms is widely recognized to be closely intertwined with their intestinal health. The intestinal mucosal layer plays a pivotal role in ensuring the proper functioning of the intestine, a fact observed not only in humans but also in animals like pigs. Any alterations to the mucosal layer of a pig's intestine can potentially disrupt its functionality, thereby impacting the animal's health and productivity. Mass spectrometry-based proteome analysis serves as a valuable tool in investigating the intricate dynamics of the proteome within the intestinal mucosa. Such studies hold promise in uncovering causal relationships between mucosal changes and overall health outcomes in pigs. It is anticipated that insights gathered from proteome studies will inform future strategies aimed at enhancing the health and productivity of pigs. However, the research field lacks a standardized and detailed method to extract proteins from pig intestinal mucosa and prepare proteins for proteome analysis. In the present study, we evaluated three alternative S-Trap-based protocols for analyzing ileal mucosal scrapings from pigs. Samples were either freeze-dried and treated as solid samples or ground in liquid nitrogen, categorized as either solid or liquid samples. In our analysis, a total of 2840 proteins were identified across all samples. Through statistical analysis and gene ontology examinations, we investigated potential differences between the three approaches. Even though our findings revealed no significant differences among the three methods, we propose the use of the protocol wherein samples are freeze-dried and treated as solid for protein extraction. This protocol stands out as the most convenient and practical option, offering ease of use and ensuring consistent and reliable results. By establishing a standardized approach, we aim to advance research efforts in understanding pig intestinal health. SignificanceThe development of an optimized protocol for protein extraction of intestinal mucosal scrapings in pigs addresses a gap in the field and enhances future research on pig intestinal health. By use of the protocol and mass spectrometry-based proteome analysis, valuable insights for improving the health and productivity of pigs can be presented. Studying the complex dynamics of the proteome within the intestinal mucosa, potentially identifying links between mucosal changes and health outcomes, provides us with information about the critical connection between intestinal health and the overall well-being and productivity of pigs. By creating a standardized approach, consistent, reliable, and reproducible results can be obtained for this type of research.

Synaptic proteome perturbations after maternal immune activation: Identification of embryonic and adult hippocampal changes

BackgroundMaternal immune activation (MIA) triggers neurobiological changes in offspring, potentially reshaping the molecular synaptic landscape, with the hippocampus being particularly vulnerable. However, critical details regarding developmental timing of these changes and whether they differ between males and females remain unclear. MethodsWe induced MIA in C57BL/6J mice on gestational day nine using the viral mimetic poly(I:C) and performed mass spectrometry-based proteomic analyses on hippocampal synaptoneurosomes of embryonic (E18) and adult (20 ± 1 weeks) MIA offspring. ResultsIn the embryonic synaptoneurosomes, MIA led to lipid, polysaccharide, and glycoprotein metabolism pathway disruptions. In the adult synaptic proteome, we observed a dynamic shift toward transmembrane trafficking, intracellular signalling cascades, including cell death and growth, and cytoskeletal organisation. In adults, many associated pathways overlapped between males and females. However, we found distinct sex-specific enrichment of dopaminergic and glutamatergic pathways. We identified 50 proteins altered by MIA in both embryonic and adult samples (28 with the same directionality), mainly involved in presynaptic structure and synaptic vesicle function. We probed human phenome-wide association study data in the cognitive and psychiatric domains, and 49 of the 50 genes encoding these proteins were significantly associated with the investigated phenotypes. ConclusionsOur data emphasise the dynamic effects of viral-like MIA on developing and mature hippocampi and provide novel targets for study following prenatal immune challenges. The 22 proteins that changed directionality from the embryonic to adult hippocampus, suggestive of compensatory over-adaptions, are particularly attractive for future investigations.

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with N in PRRSV-Infected PAMs.

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.

Characterization of Proteome Changes in Aged and Collagen VI-Deficient Human Pericyte Cultures.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

Identification of apolipoprotein E-derived amyloid within cholesterol granulomas of leopard geckos (Eublepharis macularius)

Apolipoprotein E (ApoE) is involved in cholesterol transport among cells and also plays an important role in amyloid formation, co-depositing with amyloid fibrils in various types of amyloidosis. Although the in vivo amyloidogenicity of ApoE has not been previously demonstrated, this study provides evidence of ApoE amyloidogenicity in leopard geckos (Eublepharis macularius), belonging to the class Reptilia. Histologically, amyloid deposits were localized within cholesterol granulomas and exhibited positive Congo red staining, with yellow to green birefringence under polarized light. On mass spectrometry-based proteomic analysis, ApoE was detected as a dominant component of amyloid; of the full length of the 274 amino acid residues, peptides derived from Leu185-Arg230 were frequently detected with non-tryptic truncations. Immunohistochemistry with anti-leopard gecko ApoE antibody showed positive reactions of amyloid deposits. These results show that ApoE is an amyloid precursor protein within the cholesterol granulomas of leopard geckos. Although further investigations are needed, the C-terminal region of ApoE involved in amyloid formation is a lipid-binding region, and there should be a relationship between amyloidogenesis and the development of cholesterol granulomas in leopard geckos. This study provides novel insights into the pathogenesis of ApoE-related diseases.

Systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus)

Systemic amyloid light-chain (AL) amyloidosis is an infrequent disease in which amyloid fibrils derived from the immunoglobulin light chain are deposited in systemic organs, resulting in functional impairment. This disease has been notably uncommon in animals, and nonhuman primates have not been reported to develop it. In this study, we identified the systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus) and analyzed its pathogenesis. Amyloid deposits were found severely in the submucosa of the large intestine, lung, mandibular lymph nodes, and mediastinal lymph nodes, with milder lesions in the liver and kidney. Mass spectrometry-based proteomic analysis revealed an abundant constant domain of the immunoglobulin kappa chain in the amyloid deposits. Immunohistochemistry further confirmed that the amyloid deposits were positive for immunoglobulin kappa chains. In this animal, AL amyloidosis resulted in severe involvement of the gastrointestinal submucosa and lymph nodes, which is consistent with the characteristics of AL amyloidosis in humans, suggesting that AL amyloid may have a similar deposition mechanism across species. This report enhances the pathological understanding of systemic AL amyloidosis in animals by providing a detailed characterization of this disease based on proteomic analysis.

Proteomic analysis and functional validation reveal distinct therapeutic capabilities related to priming of mesenchymal stromal/stem cells with IFN-γ and hypoxia: potential implications for their clinical use.

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Extracellular vesicles containing MFGE8 from colorectal cancer facilitate macrophage efferocytosis

BackgroundColorectal cancer (CRC) commonly exhibits tolerance to cisplatin treatment, but the underlying mechanisms remain unclear. Within the tumor microenvironment, macrophages play a role in resisting the cytotoxic effects of chemotherapy by engaging in efferocytosis to clear apoptotic cells induced by chemotherapeutic agents. The involvement of extracellular vesicles (EVs), an intercellular communicator within the tumor microenvironment, in regulating the efferocytosis for the promotion of drug resistance has not been thoroughly investigated.MethodsWe constructed GFP fluorescent-expressing CRC cell lines (including GFP-CT26 and GFP-MC38) to detect macrophage efferocytosis through flow cytometric analysis. We isolated and purified CRC-secreted EVs using a multi-step ultracentrifugation method and identified them through electron microscopy and nanoflow cytometry. Proteomic analysis was conducted to identify the protein molecules carried by CRC-EVs. MFGE8 knockout CRC cell lines were constructed using CRISPR-Cas9, and their effects were validated through in vitro and in vivo experiments using Western blotting, immunofluorescence, and flow cytometric analysis, confirming that these EVs activate the macrophage αvβ3-Src-FAK-STAT3 signaling pathway, thereby promoting efferocytosis.ResultsIn this study, we found that CRC-derived EVs (CRC-EVs) enhanced macrophage efferocytosis of cisplatin-induced apoptotic CRC cells. Analysis of The Cancer Genome Atlas (TCGA) database revealed a high expression of the efferocytosis-associated gene MFGE8 in CRC patients, suggesting a poorer prognosis. Additionally, mass spectrometry-based proteomic analysis identified a high abundance of MFGE8 protein in CRC-EVs. Utilizing CRISPR-Cas9 gene edition system, we generated MFGE8-knockout CRC cells, demonstrating that their EVs fail to upregulate macrophage efferocytosis in vitro and in vivo. Furthermore, we demonstrated that MFGE8 in CRC-EVs stimulated macrophage efferocytosis by increasing the expression of αvβ3 on the cell surface, thereby activating the intracellular Src-FAK-STAT3 signaling pathway.ConclusionsTherefore, this study highlighted a mechanism in CRC-EVs carrying MFGE8 activated the macrophage efferocytosis. This activation promoted the clearance of cisplatin-induced apoptotic CRC cells, contributing to CRC resistance against cisplatin. These findings provide novel insights into the potential synergistic application of chemotherapy drugs, EVs inhibitors, and efferocytosis antagonists for CRC treatment.

Minimal Change Disease: Pathogenetic Insights from Glomerular Proteomics.

The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers.

Proteomics study the potential targets for Rifampicin-resistant spinal tuberculosis.

Introduction: The escalating global surge in Rifampicin-resistant strains poses a formidable challenge to the worldwide campaign against tuberculosis (TB), particularly in developing countries. The frequent reports of suboptimal treatment outcomes, complications, and the absence of definitive treatment guidelines for Rifampicin-resistant spinal TB (DSTB) contribute significantly to the obstacles in its effective management. Consequently, there is an urgent need for innovative and efficacious drugs to address Rifampicin-resistant spinal tuberculosis, minimizing the duration of therapy sessions. This study aims to investigate potential targets for DSTB through comprehensive proteomic and pharmaco-transcriptomic analyses. Methods: Mass spectrometry-based proteomics analysis was employed to validate potential DSTB-related targets. PPI analysis confirmed by Immunohistochemistry (IHC) and Western blot analysis. Results: The proteomics analysis revealed 373 differentially expressed proteins (DEPs), with 137 upregulated and 236 downregulated proteins. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses delved into the DSTB-related pathways associated with these DEPs. In the context of network pharmacology analysis, five key targets-human leukocyte antigen A chain (HLAA), human leukocyte antigen C chain (HLA-C), HLA Class II Histocompatibility Antigen, DRB1 Beta Chain (HLA-DRB1), metalloproteinase 9 (MMP9), and Phospholipase C-like 1 (PLCL1)-were identified as pivotal players in pathways such as "Antigen processing and presentation" and "Phagosome," which are crucially enriched in DSTB. Moreover, pharmaco-transcriptomic analysis can confirm that 58 drug compounds can regulate the expression of the key targets. Discussion: This research confirms the presence of protein alterations during the Rifampicin-resistant process in DSTB patients, offering novel insights into the molecular mechanisms underpinning DSTB. The findings suggest a promising avenue for the development of targeted drugs to enhance the management of Rifampicin-resistant spinal tuberculosis.

Aqueous humour protein dysregulation in Asian eyes with primary open angle glaucoma

The pathophysiology of Primary Open Angle Glaucoma (POAG) remains poorly understood. Through proteomic analysis of aqueous humour (AH) from POAG patients, we aim to identify changes in protein composition of these samples compared to control samples.High resolution mass spectrometry-based TMT6plex quantitative proteomics analysis is performed on AH samples collected from POAG patients, and compared against a control group of patients with cataracts. Data are available via ProteomeXchange with identifier PXD033153.1589 proteins were quantified from the aqueous samples using Proteome Discoverer version 2.2 software. Among these proteins, 210 were identified as unique master proteins. The proteins which were up or down-regulated by ±3 fold-change were considered significant. Human neuroblastoma full-length cDNA clone CS0DD006YL02 was significantly upregulated in patients with severe POAG on >2 medications, while actin, cytoplasmic 1, V2-7 protein (fragment), immunoglobulin-like polypeptide 1 and phosphatidylethanolamine-binding protein 4 were only present in these patients with severe POAG on >2 medications.Beta-crystallin B1 and B2, Gamma-crystallin C, D and S were significantly downregulated in the severe POAG ≤2 glaucoma medications group.Beta-crystallin B2, Gamma-crystallin D and GCT-A9 light chain variable region (fragment) were significantly downregulated in the non-severe POAG group.Actin, cytoplasmic 1 was significantly upregulated in subjects with severe POAG who required more than 2 glaucoma medications. Crystallins (Beta-crystallin B1 and B2, Gamma-crystallin C, D and S) were significantly downregulated in subjects with severe POAG who required less than 2 glaucoma medications.

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck

BackgroundT cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation.MethodsWhole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells.ResultsTRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation.ConclusionsTRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

Abstract 1855: Proteomics driven biomarker discovery for high-grade serous ovarian carcinoma from archived tissue specimens

Abstract High-Grade Serous Ovarian Carcinoma (HGSOC) is a highly aggressive and heterogeneous cancer with variable treatment responses. Current treatment strategies, including surgical resection and chemotherapy, are often ineffective due to a lack of molecular markers to predict drug sensitivity and patient prognosis. Mass spectrometry-based proteomics analysis is a powerful tool for biomarker discovery, but it is restricted by the availability and quality of archived clinical specimens. In this study, we developed a proteomics-based biomarker discovery pipeline that optimizes the extraction and sample preparation from archived tissue specimens of HGSOC patients. Macrodissection of the tumor rich tissue areas of interest ensured sample purity and statistical power for comparative analysis. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we conducted a comprehensive, global proteomics analysis of FF tissue specimens from 25 HGSOC patients to associate proteomic profiles with patient outcomes. Combining high-resolution mass spectrometry proteomics of clinical samples with advanced computational tools for clinical data mining, we were able to identify pathways associated with HGSOC that hold clinical significance. Our unique biomarker discovery platform provides a novel strategy for detecting cancer vulnerabilities from archived tissue samples and identifying proteomic biomarkers linked with treatment response and patient prognosis beyond BRCA mutation status. Citation Format: Anjana Shenoy, Gali Arad, Dimitri Kovalerchik, Amit Manor, Nitzan Simchi, Dina Daitchman, Kirill Pevzner, Eran Seger. Proteomics driven biomarker discovery for high-grade serous ovarian carcinoma from archived tissue specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1855.