TMT-based Quantitative Proteomics Research Articles

TMT-based quantitative proteomics reveals the effects of electromagnetic field and freezing preservation techniques on mutton quality

This study investigated the effects of electromagnetic field preservation (EP) and freezing storage (FS) on the quality of northern Qianbei Ma mutton. Using tandem mass tagging (TMT)-labeled quantitative proteomics and bioinformatics, it was observed that EP more effectively inhibited pH increase and maintained a* and b* values compared to FS. Furthermore, the EP group was able to better maintain the water-holding capacity and tenderness of the mutton under prolonged storage. Proteomics analysis identified 397 differentially expressed proteins (DEPs) between the two storage methods at the same storage duration. GO and KEGG enrichment analyses indicated that proteins such as A0A452DSW4, A0A452E8M7, and D3JYV6 were involved in energy metabolism and redox processes, while A0A452EJ66, A0A452DSW4, and A0A452FJE8 played significant roles in protein binding. Overall, EP technology demonstrated superior benefits for maintaining mutton quality, suggesting a novel approach for mutton preservation.

ITGB4 is a prognostic biomarker and correlated with lung adenocarcinoma brain metastasis.

The aim of this study is to explore the prognostic value and immune signature of ITGB4 expression in lung adenocarcinoma (LUAD) brain metastasis. We comprehensively screened genes associated with LUAD brain metastasis by integrating datasets from the GEO database and TMT-based quantitative proteomics profiles. Univariable survival and Multivariate Cox analysis was used to compare several clinical characteristics with survival, and a risk model was constructed. The biological functions were explored via GO and KEGG analysis. Gene set enrichment analysis (GSEA) was performed using the TCGA dataset. In addition, we use TIMER to explore the collection of ITGB4 Expression and Immune Infiltration Level in LUAD. The ability of ITGB4 to regulate tumor metastasis was further assessed by migration, invasion assay and Western-blot in H1975-BrM4 cells. We found that ITGB4 was the only gene with high clinical diagnostic and prognostic value in LUAD. Enrichment analysis indicated that ITGB4 is associated with brain metastasis, infiltration of immune cells, and the response to immunotherapy. ITGB4 expression can effectively predict the outcomes of patients with LUAD who are receiving anti-PD-1 therapy. ITGB4 knockdown inhibited the invasion, migration of H1975-BrM4 brain metastasis cells, as well as epithelial-mesenchymal transition (EMT) abilities. The heightened expression of ITGB4 protein was shown to promote EMT and enhance the metastatic potential. ITGB4 promotes the progression in H1975-BrM4 cells via MEK/ERK signaling pathway. Our findings indicate that the expression of ITGB4 is linked to the occurrence of brain metastasis and infiltration of immune cells, suggesting that ITGB4 might be a clinical treatment target for LUAD.

Dynamic Cellular Proteome Remodeling during SARS-CoV-2 Infection. Identification of Plasma Protein Readouts.

The outbreak of COVID-19, led to an ongoing pandemic with devastating consequences for the global economy and human health. With the global spread of SARS-CoV-2, multidisciplinary initiatives were launched to explore new diagnostic, therapeutic, and vaccination strategies. From this perspective, proteomics could help to understand the mechanisms associated with SARS-CoV-2 infection and to identify new therapeutic options. A TMT-based quantitative proteomics and phosphoproteomics analysis was performed to study the proteome remodeling of human lung alveolar cells expressing human ACE2 (A549-ACE2) after infection with SARS-CoV-2. Detectability and the prognostic value of selected proteins was analyzed by targeted PRM. A total of 6802 proteins and 6428 phospho-sites were identified in A549-ACE2 cells after infection with SARS-CoV-2. The differential proteins here identified revealed that A549-ACE2 cells undergo a time-dependent regulation of essential processes, delineating the precise intervention of the cellular machinery by the viral proteins. From this mechanistic background and by applying machine learning modeling, 29 differential proteins were selected and detected in the serum of COVID-19 patients, 14 of which showed promising prognostic capacity. Targeting these proteins and the protein kinases responsible for the reported phosphorylation changes may provide efficient alternative strategies for the clinical management of COVID-19.

TMT-based quantitative proteomics analysis of defense responses induced by the Bph3 gene following brown planthopper infection in rice.

The brown planthopper (BPH) is an economically significant pest of rice. Bph3 is a key BPH resistance gene. However, the proteomic response of rice to BPH infestation, both in the presence and absence of Bph3, remains largely unexplored. In this study, we employed tandem mass tag labeling in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins (DEPs) in rice samples. We detected 265 and 125 DEPs via comparison of samples infected with BPH for 2 and 4 days with untreated samples of the BPH-sensitive line R582. For the Bph3 introgression line R373, we identified 29 and 94 DEPs in the same comparisons. Bioinformatic analysis revealed that Bph3 significantly influences the abundance of proteins associated with metabolic pathways, secondary metabolite biosynthesis, microbial metabolism in diverse environments, and phenylpropanoid biosynthesis. Moreover, Bph3 regulates the activity of proteins involved in the calcium signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction. Our results indicate that Bph3 enhances the resistance of rice to BPH mainly by inhibiting the down-regulation of proteins associated with metabolic pathways; calcium signaling, the MAPK signaling pathway, and plant hormone signal transduction might also be involved in BPH resistance induced by Bph3.

The modulation of proteomics and antioxidant stress is involved in the effect of nitazoxanide against Japanese encephalitis virus in vitro

Japanese encephalitis virus (JEV) is a significant circulating arbovirus flavivirus and the primary cause of viral encephalitis in Asia. Previous studies have demonstrated that nitazoxanide (NTZ), an antiparasitic gastroenteritis medication classified as a thiazolide, exhibits efficacy against JEV both in vitro and in vivo. To explore the potential antiviral mechanisms, we employed Tandem Mass Tag (TMT)-based quantitative proteomics to identify differentially expressed proteins (DEPs) among three groups: Blank cell group, JEV-infected cell group, and JEV-infected cells treated with NTZ. Our analysis revealed that NTZ treatment led to the upregulation of 30 DEPs and downregulation of 54 DEPs in JEV-infected cells. Enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these DEPs are involved in various biological processes and signaling pathways, including transport, localization, response to wounding, P53 pathway activation, and fatty acid metabolism-related pathways. Moreover, we observed that the expression trend of TMX2, a protein associated with redox homeostasis, was consistent with findings from TMT-based quantitative proteomics. Further investigations into reactive oxygen species (ROS), mitochondrial membrane potential, antioxidant enzyme activity, and the KEAP1-NRF2 pathway demonstrated that NTZ effectively regulates the KEAP1-NRF2 pathway while suppressing oxidative stress induced by JEV infection. In conclusion, the proteomic data along with antioxidant stress results presented herein provide a foundational basis for further research into the molecular mechanisms and potential targets of NTZ against JEV.

SPOT: spatial proteomics through on-site tissue-protein-labeling

BackgroundSpatial proteomics seeks to understand the spatial organization of proteins in tissues or at different subcellular localization in their native environment. However, capturing the spatial organization of proteins is challenging. Here, we present an innovative approach termed Spatial Proteomics through On-site Tissue-protein-labeling (SPOT), which combines the direct labeling of tissue proteins in situ on a slide and quantitative mass spectrometry for the profiling of spatially-resolved proteomics.Materials and MethodsEfficacy of direct TMT labeling was investigated using seven types of sagittal mouse brain slides, including frozen tissues without staining, formalin-fixed paraffin-embedded (FFPE) tissues without staining, deparaffinized FFPE tissues, deparaffinized and decrosslinked FFPE tissues, and tissues with hematoxylin & eosin (H&E) staining, hematoxylin (H) staining, eosin (E) staining. The ability of SPOT to profile proteomes at a spatial resolution was further evaluated on a horizontal mouse brain slide with direct TMT labeling at eight different mouse brain regions. Finally, SPOT was applied to human prostate cancer tissues as well as a tissue microarray (TMA), where TMT tags were meticulously applied to confined regions based on the pathological annotations. After on-site direct tissue-protein-labeling, tissues were scraped off the slides and subject to standard TMT-based quantitative proteomics analysis.ResultsTissue proteins on different types of mouse brain slides could be directly labeled with TMT tags. Moreover, the versatility of our direct-labeling approach extended to discerning specific mouse brain regions based on quantitative outcomes. The SPOT was further applied on both frozen tissues on slides and FFPE tissues on TMAs from prostate cancer tissues, where a distinct proteomic profile was observed among the regions with different Gleason scores.ConclusionsSPOT is a robust and versatile technique that allows comprehensive profiling of spatially-resolved proteomics across diverse types of tissue slides to advance our understanding of intricate molecular landscapes.

Experimental and proteomics evidence revealed the protective mechanisms of Shemazhichuan Liquid in attenuating neutrophilic asthma

BackgroundNeutrophilic asthma (NA) is one of the most important phenotypes of non-Th2 asthma and is often insensitive to glucocorticoid therapy, making current treatment difficult. Shemazhichuan Liquid (SMZCL), a Chinese medicine compound preparation, has unique advantages in the treatment of asthma. However, the underlying mechanisms of SMZCL in treating NA are not fully understood. PurposeThe efficacy and underlying mechanisms of SMZCL on NA were investigated by TMT-labeled quantitative proteomics analysis and in vivo and in vitro experiments. MethodsNA mouse model was constructed by OVA/CFA sensitization followed by a 10-day challenge with 5 % OVA. Lung histopathology, leukocyte counts and cell sorting counts, inflammatory cytokines levels, as well as expression of autophagy markers were then assessed. The specific pathways and proteins of SMZCL for treating NA were further illustrated through TMT-based quantitative proteomics. In addition, RAW264.7 cells were induced by LPS to further explore the mechanism of the main active ingredient of SMZCL on autophagy pathway. ResultsIn vivo, SMZCL contributed to attenuating airway inflammation and collagen disposition, markedly reduced the number of leukocytes, especially neutrophils in bronchoalveolar lavage fluid (BALF), as well as decreased IgE and inflammatory cytokine levels (TNF-α, IL-1β, IL-6 and IL-8) in BALF and serum. Besides, SMZCL elevated the levels of LC3 and ATG5 while inhibiting the expression of p62 and mTOR. Mzb1 and Rab3ip were identified as the critical overlapping DEPs whose expression was inhibited by SMZCL and rapamycin. KEGG enrichment analysis showed that necroptosis process was a key pathway for SMZCL to treat NA airway inflammation. IHC and WB results confirmed that SMZCL and rapamycin inhibited the phosphorylation of RIPK1, PIPK3 and MLKL. In vitro, ATG5 and LC3 proteins were obviously increased while p-mTOR expression was inhibited after amygdalin treatment. ConclusionSMZCL attenuated airway inflammation in NA mainly through inhibition of the mTOR pathway, along with inhibition of the necroptosis pathway regulated by the RIPK1/RIPK3/MLKL axis and inhibition of Mzb1 and Rab3ip expression.

TMT-based quantitative proteomics unveils the protective mechanism of Polygonatum sibiricum polysaccharides on septic acute liver injury

Polygonatum sibiricum polysaccharides (PSP) has been shown to possess multiple pharmacological functions. Our previous study found that PSP could protect against acute liver injury during sepsis via inhibiting inflammatory response. However, the underlying molecular mechanism by which PSP alleviates septic acute liver injury (SALI) remains unknown. Herein, TMT-based quantitative proteomics was utilized to explore the essential pathways and proteins involved in the protective effects of PSP on SALI. The results revealed that 632 and 176 differentially expressed proteins (DEPs) were identified in Model_vs_Control and PSP_vs_Model, respectively. GO annotation showed similar trends, suggesting that these DEPs were primarily involved in the cellular anatomical entity in Cellular Component, the cellular processe and the biological regulation in Biological Process, the binding and the catalytic activity in Molecular Function. Meanwhile, KEGG enrichment analysis implied that four common pathways, including the NF-κB signaling pathway, the IL-17 signaling pathway, the TNF signaling pathway and the Toll-like receptor signaling pathway, were closely associated with the pathogenesis of sepsis among the top 20 remarkably enriched pathways in Model_vs_Control_up and PSP_vs_Model_down. Moreover, the levels of several common DEPs, including TLR2, IKKi, JunB and CXCL9, were validated by WB, which was in line with the results of proteomics. Therefore, the protective effects of PSP on SALI might exert via blocking the above-mentioned inflammation pathways.Significance: PSP, recognized as a key component of Polygonatum sibiricum, exhibits a range of pharmacological functions. Our previous study found that PSP could protect against SALI, yet failing to clarify the mechanism of action. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP. SignificancePSP have been identified as the most crucial components of Polygonatum sibiricum with various pharmacological functions. Our previous study found that PSP could protect against SALI, but the mechanism of action remains unknown. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP.

TMT-Based Quantitative Proteomics Revealed the Antibacterial Mechanism of Cinnamaldehyde against MRSA.

Natural plant extracts have demonstrated significant potential in alternative antibiotic therapies. Cinnamaldehyde (CA) has garnered considerable attention as a natural antibacterial agent. In this study, Tandem mass tag (TMT) quantitative proteomics combined with Western blot and RT-qPCR methods were employed to explore the antibacterial mechanism of CA against Methicillin-Resistant Staphylococcus aureus (MRSA) at the protein level. The results showed that a total of 254 differentially expressed proteins (DEPs) were identified in the control group and CA treatment group, of which 161 were significantly upregulated and 93 were significantly downregulated. DEPs related to nucleotide synthesis, homeostasis of the internal environment, and protein biosynthesis were significantly upregulated, while DEPs involved in the cell wall, cell membrane, and virulence factors were significantly downregulated. The results of GO and KEGG enrichment analyses demonstrated that CA could exert its antibacterial effects by influencing pyruvate metabolism, the tricarboxylic acid (TCA) cycle, teichoic acid biosynthesis, and the Staphylococcus aureus (S. aureus) infection pathway in MRSA. CA significantly inhibited the expression of recombinant protein MgrA (p < 0.05), significantly reduced the mRNA transcription levels of mgrA, hla, and sdrD genes (p < 0.05), and thermostability migration assays demonstrated that CA can directly interact with MgrA protein, thereby inhibiting its activity. These findings suggest that CA exerts its antibacterial mechanism by regulating the expression of related proteins, providing a theoretical basis for further development of clinical applications of antimicrobial agents derived from natural plant essential oils in the treatment of dairy cow mastitis.

The Low-Abundance Plasma Proteome Reveals Differentially Abundant Proteins Associated with Breast Implant Capsular Contracture: A Pilot Study.

Capsular contracture (CC) is one of the most common postoperative complications associated with breast implant-associated infections. The mechanisms that lead to CC remain poorly understood. Plasma is an ideal biospecimen for early proteomics biomarker discovery. However, as high-abundance proteins mask signals from low-abundance proteins, identifying novel or specific proteins as biomarkers for a particular disease has been hampered. Here, we employed depletion of high-abundance plasma proteins followed by Tandem Mass Tag (TMT)-based quantitative proteomics to compare 10 healthy control patients against 10 breast implant CC patients. A total of 450 proteins were identified from these samples. Among them, 16 proteins were significantly differentially expressed in which 5 proteins were upregulated and 11 downregulated in breast implant CC patients compared to healthy controls. Gene Ontology enrichment analysis revealed that proteins related to cell, cellular processes and catalytic activity were highest in the cellular component, biological process, and molecular function categories, respectively. Further, pathway analysis revealed that inflammatory responses, focal adhesion, platelet activation, and complement and coagulation cascades were enriched pathways. The differentially abundant proteins from TMT-based quantitative proteomics have the potential to provide important information for future mechanistic studies and in the development of breast implant CC biomarkers.

TMT-based quantitative proteomics and non-targeted metabolomic analyses reveal the inactivation mechanism of cold atmospheric plasma against Pseudomonas aeruginosa

Cold atmospheric plasma (CAP) has emerged as a potent nonthermal inactivation strategy in food processing over the past two decades. However, the underlying mechanisms of inactivation remain unclear. In this study, Tandem Mass Tag (TMT)-based quantitative proteomics and non-targeted metabolomic analyses were conducted to investigate the responses of Pseudomonas aeruginosa to CAP. The results revealed significant alterations in 170 differentially expressed proteins (DEPs) and 490 differential metabolites (DMs) upon air-CAP treatment. P. aeruginosa demonstrated a regulatory response at the transcription and translation levels, upregulating proteins to resist external stimuli. Conversely, a predominant down-regulation of proteins indicated that CAP treatment profoundly disrupted the cell structure, inhibiting movement, colonization ability, virulence protein secretion, and bacterial biofilm formation. Moreover, CAP compromised the bacterium's ability to acquire energy, thereby disrupting its defense mechanisms, reducing drug resistance, and potentially leading to bacterial death. This comprehensive study enhances our understanding of the mode of action of air-CAP against P. aeruginosa. The findings provide a robust experimental foundation for considering CAP as an effective inactivation method in the food industry.

Podophyllotoxin via SIRT1/PPAR /NF-κB axis induced cardiac injury in rats based on the toxicological evidence chain (TEC) concept

BackgroundThe study of cardiotoxicity of drugs has become an important part of clinical safety evaluation of drugs. It is commonly known that podophyllotoxin (PPT) and its many derivatives and congeners are broad-spectrum pharmacologically active substances. Clinical cardiotoxicity of PPT and its derivatives has been raised, basic research on the mechanism of cardiotoxicity remains insufficient. PurposeIn present study, our group's innovative concept of toxicological evidence chain (TEC) was applied to reveal the cardiac toxicity mechanism of PPT by targeted metabolomics, TMT-based quantitative proteomics and western blot. MethodsThe injury phenotype evidence (IPE) acquired from the toxicity manifestations, such as weight and behavior observation of Sprague-Dawley rat. The damage to rat hearts were assessed through histopathological examination and myocardial enzymes levels, which were defined as Adverse Outcomes Evidence (AOE). The damage to rat hearts was assessed through histopathological examination and myocardial enzyme levels, which were defined as evidence of adverse outcomes.Overall measurements of targeted metabolomics based on energy metabolism and TMT-based quantitative proteomics were obtained after exposure to PPT to acquire the Toxic Event Evidence (TEE). The mechanism of cardiac toxicity was speculated based on the integrated analysis of targeted metabolomics and TMT-based quantitative proteomics, which was verified by western blot. ResultsThe results indicated that exposure to PPT could result in significant elevation of myocardial enzymes and pathological alterations in rat hearts. In addition, we found that PPT caused disorders in cardiac energy metabolism, characterized by a decrease in energy metabolism fuels. TMT-based quantitative proteomics revealed that the PPAR (Peroxisome proliferators-activated receptor) signaling pathway needs further study. It is worth noting that PPT may suppress the expression of SIRT1, subsequently inhibiting AMPK, decreasing the expression of PGC-1α, PPARα and PPARγ. This results in disorders of glucose oxidation, glycolysis and ketone body metabolism. Additionally, the increase in the expression of p-IKK and p-IκBα, leads to the nuclear translocation of NF-κB p65 from the cytosol, thus triggering inflammation. ConclusionThis study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of cardiotoxicity,suggesting that PPT induced disorders of energy metabolism and inflammation via SIRT1/PPAR/NF-κB axis, potentially contributing to cardiac injury.

Integrated transcriptome and proteome analysis reveals the unique molecular features and nutritional components on the muscles in Chinese Taihe black-bone silky fowl chicken.

The Taihe Black-Bone silky fowl chicken (BB-sfc) is a renowned dietary and medicinal chicken globally recognized for its high nutritional and medicinal value. Compared to the local Black-Bone black-feathered chicken (BB-bfc), the Taihe silky fowl chicken has higher levels of amino acids, trace elements, and unsaturated fatty acids in their muscles, which offer anti-aging, anti-cancer, and immune enhancing benefits. Despite this, the unique nutritional components, genes, and proteins in Taihe silky fowl chicken muscles are largely unknown. Therefore, we performed a comprehensive transcriptome and proteome analysis of muscle development between BB-sfc and BB-bfc chickens using RNA-Seq and TMT-based quantitative proteomics methods. RNA-Seq analysis identified 286 up-regulated genes and 190 down-regulated genes in BB-sfc chickens, with oxidoreductase activity and electron transfer activity enriched in up-regulated genes, and phospholipid homeostasis and cholesterol transporter activity enriched in down-regulated genes. Proteome analysis revealed 186 significantly increased and 287 significantly decreased proteins in Taihe BB-sfc chicken muscles, primarily affecting mitochondrial function and oxidative phosphorylation, crucial for enhancing muscle antioxidant capacity. Integrated transcriptome and proteome analysis identified 6 overlapped up-regulated genes and 8 overlapped down-regulated genes in Taihe silky fowl chicken, related to improved muscle antioxidant status. Taken together, this research provides a comprehensive database of gene expression and protein information in Taihe Black-Bone silky fowl chicken muscles, aiding in fully exploring their unique economic value in the future.

Proteomic analysis of plasma proteins from patients with cardiac rupture after acute myocardial infarction using TMT-based quantitative proteomics approach

BackgroundCardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of CR has yet to be elucidated.MethodsIn the present study, a quantitative approach with tandem mass tag (TMT) labeling and liquid chromatography–tandem mass spectrometry was used to characterize the differential protein expression profiles of patients with CR. Plasma samples were collected from patients with CR (n = 37), patients with AMI (n = 47), and healthy controls (n = 47). Candidate proteins were selected for validation by multiple reaction monitoring (MRM) and enzyme-linked immunosorbent assay (ELISA).ResultsIn total, 1208 proteins were quantified and 958 differentially expressed proteins (DEPs) were identified. The difference in the expression levels of the DEPs was more noticeable between the CR and Con groups than between the AMI and Con groups. Bioinformatics analysis showed most of the DEPs to be involved in numerous crucial biological processes and signaling pathways, such as RNA transport, ribosome, proteasome, and protein processing in the endoplasmic reticulum, as well as necroptosis and leukocyte transendothelial migration, which might play essential roles in the complex pathological processes associated with CR. MRM analysis confirmed the accuracy of the proteomic analysis results. Four proteins i.e., C-reactive protein (CRP), heat shock protein beta-1 (HSPB1), vinculin (VINC) and growth/differentiation factor 15 (GDF15), were further validated via ELISA. By receiver operating characteristic (ROC) analysis, combinations of these four proteins distinguished CR patients from AMI patients with a high area under the curve (AUC) value (0.895, 95% CI, 0.802–0.988, p < 0.001).ConclusionsOur study highlights the value of comprehensive proteomic characterization for identifying plasma proteome changes in patients with CR. This pilot study could serve as a valid foundation and initiation point for elucidation of the mechanisms of CR, which might aid in identifying effective diagnostic biomarkers in the future.

TMT-based quantitative proteomics analysis of Sprague-Dawley rats liver reveals Triphenyltin induced liver damage and lipid metabolism disorders

Triphenyltin (TPT) is a widely used pesticide that has a negative impact on biological health and production efficiency. In addition, TPT poses a threat to human health through the food chain and environmental pollution. However, the exact mechanism of TPT toxicity remains unclear. In this study, we investigated the hepatotoxicity of TPT and its effects on lipid metabolism using male SD rats as an animal model. Our results from HE and serum biochemical analysis suggested that TPT could damage liver structure and function, resulting in disruption of lipid metabolism. We therefore proceeded to analyze the proteomic response of rat liver tissue after 28 days of treatment with 2 mg/kg/d TPT. Our study demonstrates that TPT has a variety of effects on liver protein expression in rats. Through bioinformatic analysis, we observed significant changes in proteins related to fatty acid oxidation and synthesis due to TPT exposure. Furthermore, western blot and RT-qPCR experiments confirmed that TPT can affect lipid metabolism through the PPAR pathway. These findings suggest that TPT exposure can lead to liver damage, lipid accumulation and metabolic disorders.

TMT-based quantitative proteomics analysis of neuroprotective effects of Forsythoside A on the MPTP-induced Parkinson's disease mouse model

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characteristized by the presence of dyskinesia and the progressive loss of dopaminergic neurons. Although certain drugs can mitigate the symptoms of PD, they are unable to delay the disease progression, and their prolonged use may result in complications. Therefore, there exists an urgent necessity to identify potential agents that can effectively delay PD progression with fewer side effects. Recent research has unveiled that several traditional Chinese medicines (TCM) exhibit neuroprotective properties in various models pertinent to PD. Forsythoside A (FSA), the primary bioactive compound derived from TCM Lianqiao, has undergone extensive research in animal models of Alzheimer's disease and cerebral ischemia. However, the investigation into the impact of FSA on PD is limited in existing research. In this study, we aimed to evaluate the neuroprotective effects of FSA on MPTP-induced PD mouse model. FSA demonstrated significant improvements in the behavioral and neuropathological changes triggered by MPTP in mice. Furthermore, it exerted a suppressive effect on the activations of astrocyte and microglia. Meanwhile, Tandem mass tag (TMT)-based quantitative proteomics of striatal tissue and bioinformatics analysis were performed to elucidate the underlying mechanisms of FSA on PD mouse model. Proteomics demonstrated a total of 68 differentially expressed proteins (DEPs) were identified between HFSA and MPTP groups including 26 upregulated and 42 downregulated. Systematic bioinformatics analysis of the 68 DEPs illustrated that they were predominantly related to estrogen signaling pathway and calcium signaling pathway. The related DEPs (PLCβ4, Grm2, HPAC and Cox4i1) expression levels were verified by Western blot. FSA effectively restored the altered expression of the four DEPs induced by MPTP. Summarily, FSA exerted remarkable neuroprotective effects in MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of FSA as a potential treatment for PD.

TMT-based quantitative proteomics reveals the nutritional and stress resistance functions of anaerobic fungi in yak rumen during passage at different time intervals

ObjectivesAnaerobic fungi are critical for nutrient digestion in the yak rumen. Although studies have reported the effects of passage at different time intervals on the community structure of yak rumen anaerobic fungi, it is unknown whether passage culture at different time intervals affects the microbial proteins of rumen anaerobic fungi and their functions. MethodsMycelium was obtained using the anaerobic continuous batch culture (CBC) of yak rumen fluid at intervals of 3 d, 5 d and 7 d. Quantitative analysis of fungal proteins and functional analysis was performed using tandem mass tagging (TMT) and bioinformatics. ResultsA total of 56 differential proteins (DPs) were found in 5 d vs. 3 d and 7 d vs. 3 d. Gene ontology (GO) enrichment indicated that the up-regulated proteins were mainly involved in biological regulation, cellular process, metabolic process, macromolecular complex, membrane, cell part, organelle, binding, catalytic activity and transporter activity. The downregulated proteins were mainly enriched in metabolic process, cell part, binding and catalytic activity. Furthermore, the downregulated proteins in 7 d vs. 3 d were related to membrane and organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicated that DPs were enriched in 14 pathways in 5 d vs. 3 d and 7 d vs. 3 d, mainly including terpenoid backbone biosynthesis, alaine, aspartate and glutamate metabolism, arginine biosynthesis, hypotaurine, cyanoamino acid, glutathione, β-alanine, pyrimidine, purine, galactose and propanate metabolism, steroid biosynthesis, ribosome biogenesis in eukaryotes and aminoacyl tRNA biosynthesis. The DPs were enriched in only 2 pathways in 5 d vs 3 d, lysine biosynthesis and cysteine and methionine metabolism. N-glycan biosynthesis and retinol metabolism are only found in the metabolism of DPs in 7 d vs 3 d. ConclusionsYak rumen anaerobic fungal proteins are involved in nutrition and stress tolerance during passage at different time intervals.

TMT-Based Proteomics Reveal the Mechanism of Action of Amygdalin against Rheumatoid Arthritis in a Rat Model through Regulation of Complement and Coagulation Cascades.

The limitations of current medications for treating rheumatoid arthritis (RA) emphasize the urgent need for the development of new drugs. This study aimed to investigate the potential anti-RA mechanism of amygdalin using tandem mass tag (TMT)-based quantitative proteomics technology. First, the anti-RA activity of amygdalin was evaluated in a Complete Freund's adjuvant (CFA)-induced rat model. Then, the roles and importance of proteins in the extracted rat joint tissue were evaluated using TMT-based quantitative proteomics technology. A bioinformatics analysis was used to analyze differentially abundant proteins (DAPs). A proteomics analysis identified 297 DAPs in the amygdalin group compared with the model group, of which 53 upregulated proteins and 51 downregulated proteins showed opposite regulatory trends to the DAPs produced after modeling. According to enrichment analyses of the DAPs, the signaling pathways with a high correlation degree were determined to be the complement and coagulation cascades. Furthermore, western blotting and molecular docking were used to further validate the key node proteins, e.g., complement C1s subcomponent (C1s), component C3 (C3) and kininogen 1 (Kng1). These results suggest that amygdalin may be a promising agent for treating RA by regulating the complement and coagulation cascades.

TMT-based quantitative proteomics reveals the targets of andrographolide on LPS-induced liver injury

BackgroundAndrographolide (Andro) is a diterpenoid derived from Andrographis paniculate, which has anti-inflammatory, antibacterial, antiviral and hepatoprotective activities. Gram-negative bacterial infections can cause varying degrees of liver injury in chickens, although Andro has been shown to have a protective effect on the liver, its underlying mechanism of action and effects on liver proteins are not known.MethodsThe toxicity of Andro on the viability of leghorn male hepatoma (LMH) cells at different concentrations and times was analyzed by CCK-8 assays. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the culture supernatants were measured using an automatic biochemical analyzer to evaluate the protective effect of androscopolide on LPS-induced injury of LMH cells. Subsequently, TMT proteomics analysis were performed on the negative control group (NC group), LPS, and LPS-Andro groups, and bioinformatics analysis was performed on the differentially expressed proteins (DEPs).ResultsIt was found that Andro reduced ALT and AST levels in the cell supernatant and alleviated LPS-induced injury in LMH cells. Proteomic analysis identified 50 and 166 differentially expressed proteins in the LPS vs. NC group and LPS-Andro vs. LPS group, respectively. Andro may be involved in steroid metabolic processes, negative regulation of MAPK cascade, oxidative stress, and other processes to protect against LPS-induced liver injury.ConclusionsAndro protects against LPS-induced liver injury, HMGCS1, HMGCR, FDPS, PBK, CAV1, PRDX1, PRDX4, and PRDX6, which were identified by differential proteomics, may be the targets of Andro. Our study may provide new theoretical support for Andro protection against liver injury.

TMT-based quantitative proteomics reveals the protective mechanism of tenuigenin after experimental intracerebral hemorrhage in mice

Ethnopharmacological relevanceTenuigenin (TNG) is an extract obtained from Polygalae Radix. It possesses anti-inflammatory, antioxidant, and neuroprotective properties. However, the potential mechanism of TNG in intracerebral hemorrhage (ICH) has not been well studied. Aim of the studyIn the present study, we aimed to identify the prospective mechanism of TNG in treating ICH. Materials and methodsA total of 120 mice were divided into five groups: Sham group, ICH + vehicle group, ICH + TNG(8 mg/kg), ICH + TNG(16 mg/kg), and ICH + TNG(32 mg/kg). The modified Garcia test and beam walking test were carried out at 24 h and 72 h after ICH. Brain water content, haematoma volume and hemoglobin content examinations were performed at 72 h after ICH. TMT-based quantitative proteomics combined with bioinformatics analysis methods was used to distinguish differentially expressed proteins (DEPs) to explore potential pharmacological mechanisms. Western blotting was performed to validate representative proteins. ResultsOur results showed that the optimal dose of TNG was 16 mg/kg, which could markedly improve neurological functions, and reduce cerebral oedema, haematoma volume and hemoglobin levels 72 h after ICH. A total of 404 DEPs (353 up-and 51 downregulated) were identified in the ICH + vehicle vs. sham group, while 342 DEPs (306 up-and 36 downregulated) and 76 DEPs (28 up-and 48 downregulated) were quantified in the TNG vs. sham group and TNG vs. ICH + vehicle group, respectively. In addition, a total of 26 DEPs were selected according to strict criteria. Complement and coagulation cascades were the most significantly enriched pathways, and two proteins (MBL-C and Car1) were further validated as hub molecules. ConclusionsOur results suggested that the therapeutic effects of TNG on ICH were closely associated with the complement system, and that MBL-C and Car1 might be potential targets of TNG for the treatment of ICH.