Tandem Mass Tag-based Proteomics Research Articles

Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics.

Isobaric labeling is widely used for unbiased, proteome-wide studies, and it provides several advantages, such as fewer missing values among samples and higher quantitative precision. However, ion interference may lead to compressed or distorted observed ratios due to the coelution and coanalysis of peptides. Here, we introduced a synthetic KnockOut standard (sKO) for evaluating interference in tandem mass tags-based proteomics. sKO is made by mixing TMTpro-labeled tryptic peptides derived from four nonhuman proteins and a whole human proteome as background at different proportions. We showcased the utility of the sKO standard by exploring ion interference at different peptide concentrations (up to a 30-fold change in abundance) and using a variety of mass spectrometer data acquisition strategies. We also demonstrated that the sKO standard could provide valuable information for the rational design of acquisition strategies to achieve optimal data quality and discussed its potential applications for high-throughput proteomics workflows development.

Schistosoma mansoni infection induces hepatic metallothionein and S100 protein expression alongside metabolic dysfunction in hamsters.

Schistosomiasis, a widespread neglected tropical disease, presents a complex and multifaceted clinical-pathological profile. Using hamsters as final hosts, we dissected molecular events following Schistosoma mansoni infection in the liver-the organ most severely affected in schistosomiasis patients. Employing tandem mass tag-based proteomics, we studied alterations in the liver proteins in response to various infection modes and genders. We examined livers from female and male hamsters that were: noninfected (control), infected with either unisexual S. mansoni cercariae (single-sex) or both sexes (bisex). The infection induced up-regulation of proteins associated with immune response, cytoskeletal reorganization, and apoptotic signaling. Notably, S. mansoni egg deposition led to the down-regulation of liver factors linked to energy supply and metabolic processes. Gender-specific responses were observed, with male hamsters showing higher susceptibility, supported by more differentially expressed proteins than found in females. Of note, metallothionein-2 and S100a6 proteins exhibited substantial up-regulation in livers of both genders, suggesting their pivotal roles in the liver's injury response. Immunohistochemistry and real-time-qPCR confirmed strong up-regulation of metallothionein-2 expression in the cytoplasm and nucleus upon the infection. Similar findings were seen for S100a6, which localized around granulomas and portal tracts. We also observed perturbations in metabolic pathways, including down-regulation of enzymes involved in xenobiotic biotransformation, cellular energy metabolism, and lipid modulation. Furthermore, lipidomic analyses through liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry imaging identified extensive alterations, notably in cardiolipin and triacylglycerols, suggesting specific roles of lipids during pathogenesis. These findings provide unprecedented insights into the hepatic response to S. mansoni infection, shedding light on the complexity of liver pathology in this disease.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

Postharvest senescence profiling in broccoli using Tandem Mass Tag-based proteomics

Postharvest senescence profiling in broccoli using Tandem Mass Tag-based proteomics

Automated Sample Preparation Workflow for Tandem Mass Tag-Based Proteomics.

Although tandem mass tag (TMT)-based isobaric labeling has become a powerful approach for multiplexed protein quantitation, automating the workflow for this technique has not been easy to achieve for widespread adoption. This is because preparation of TMT-labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction, and alkylation to tryptic digestion, desalting, labeling, and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic, especially with large-scale clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for an automated proteomic workflow employing TMT labeling with the manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using an automated platform and a conventional manual protocol. Single-shot liquid chromatography with tandem mass spectrometry analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiency of tryptic digestion, alkylation, and TMT labeling were similar in both manual and automated processes. In addition, comparison of quantitation accuracy and precision showed similar performance in an automated workflow compared to manual sample preparation by an expert. Overall, we demonstrated that the automated platform performs at a level similar to a manual process performed by an expert for TMT-based proteomics. We anticipate that this automated workflow will increasingly replace manual pipelines and has the potential to be applied to large-scale TMT-based studies, providing robust results and high sample throughput.

Tandem mass tag-based proteomics technology provides insights into multi-targeted mechanism of peptide MOp2 from Moringa oleifera seeds against Staphylococcus aureus

Our previous study proved that the peptide MOp2 from Moringa oleifera seeds exhibited membrane-damaging effects on Staphylococcus aureus. In this study, TMT-based proteomics technology was mainly used to investigate the antimicrobial mechanism of MOp2 against S. aureus. A total of 541 differentially expressed proteins (DEPs) were identified in S. aureus treated with MOp2, among which 256 and 285 DEPs were upregulated and downregulated, respectively. They were mainly involved in inositol phosphate metabolism, S. aureus infection, citrate cycle (TCA cycle), and phosphotransferase system (PTS) and acted as ABC transporters and ribosomes. Moreover, the decreasing AKP activity indicated that MOp2 affected cell wall biosynthesis, and excessive ROS accumulation caused apoptosis, resulting in DNA fragmentation, nuclear morphological changes, and phosphatidylserine externalization. Additionally, molecular docking showed that MOp2 could interact with eight DEPs, including ProC, QoxB, SOD2, DnaK, GroEL, RplY, AcpS, and FabG, indicating that MOp2 might act on these molecular targets, leading to increased cell permeability, oxidative damage, impaired protein synthesis, cell wall synthesis obstruction, and energy metabolism disorder. Overall, these findings provided new insights into the multi-targeted mechanism of MOp2 against S. aureus, and could provide a theoretical basis for its application as a novel food preservative and antibiotic substitute.

Molecular mechanism of Cuscutae semen-radix rehmanniae praeparata in relieving reproductive injury of male rats induced with tripterygium wilfordii multiglycosides: A tandem mass tag-based proteomics analysis.

Background: We determined the effects of Cuscutae semen (Cuscuta chinensis Lam. or Cuscuta australis R. Br.)-Radix rehmanniae praeparata (Rehjnannia glutinosa Libosch.) on the protein levels in testicular tissues of rats gavaged with tripterygium wilfordii multiglycosides (GTW) and elucidated the molecular mechanism underlying Cuscutae semen-Radix rehmanniae praeparata for relieving GTW-induced reproductive injury. Methods: A total of 21 male Sprague-Dawley rats were randomly divided into the control group, model group, and Cuscutae semen-Radix rehmanniae praeparata group based on their body weights. The control group was given 10mLkg-1 of 0.9% normal saline by gavage daily. The model group (GTW group) was administered with 12mgkg-1 GTW by gavage daily. Cuscutae semen-Radix rehmanniae praeparata group (the TSZSDH group) was administered with 1.56gkg-1 of Cuscutae semen-Radix rehmanniae praeparata granules daily according to their model group dosing. The serum levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and testosterone were measured after 12weeks of continuous gavage, and the pathological lesion of testicular tissues was observed. Differentially expressed proteins were evaluated by quantitative proteomics and verified by western blotting (WB) and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Results: Cuscutae semen-Radix rehmanniae praeparata can effectively relieve pathological lesions of GTW-induced testicular tissues. A total of 216 differentially expressed proteins were identified in the TSZSDH group and model group. High-throughput proteomics revealed that differentially expressed proteins are closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and protein glycan pathway in cancer. Cuscutae semen-Radix rehmanniae praeparata can significantly upregulate the protein expressions of Acsl1, Plin1, Dbil5, Plin4, Col12a1, Col1a1, Col5a3, Col1a2, Dcn, so as to play a protective role on testicular tissues. Acsl1, Plin1, and PPARγ on the PPAR signaling pathway were verified by WB and RT-qPCR experiments, which were found to be consistent with the results of proteomics analysis. Conclusion: Cuscutae semen and Radix rehmanniae praeparata may regulate the PPAR signaling pathway mediated Acsl1, Plin1 and PPARγ to reduce the testicular tissue damage of male rats caused by GTW.

Integrative analysis and identification of key elements and pathways regulated by Traditional Chinese Medicine (Yiqi Sanjie formula) in colorectal cancer.

Introduction: The clinical efficacy of Yiqi Sanjie (YQSJ) formula in the treatment of stage III colorectal cancer (CRC) has been demonstrated. However, the underlying antitumor mechanisms remain poorly understood. Materials and methods: The aim of the present study was to comprehensively characterize the molecular and microbiota changes in colon tissues and fecal samples from CRC mice and in CRC cell lines treated with YQSJ or its main active component, peiminine. Integrative tandem mass tag-based proteomics and ultra-performance liquid chromatography coupled with time-of-flight tandem mass spectrometry metabolomics were used to analyze azoxymethane/dextran sulfate sodium-induced CRC mouse colon tissues. Results: The results showed that 0.8% (57/7568) of all detected tissue proteins and 3.2% (37/1141) of all detected tissue metabolites were significantly changed by YQSJ treatment, with enrichment in ten and six pathways associated with colon proteins and metabolites, respectively. The enriched pathways were related to inflammation, sphingolipid metabolism, and cholesterol metabolism. Metabolomics analysis of fecal samples from YQSJ-treated mice identified 121 altered fecal metabolites and seven enriched pathways including protein digestion and absorption pathway. 16S rRNA sequencing analysis of fecal samples indicated that YQSJ restored the CRC mouse microbiota structure by increasing the levels of beneficial bacteria such as Ruminococcus_1 and Prevotellaceae_UCG_001. In HCT-116 cells treated with peiminine, data-independent acquisition-based proteomics analysis showed that 1073 of the 7152 identified proteins were significantly altered and involved in 33 pathways including DNA damage repair, ferroptosis, and TGF-β signaling. Conclusion: The present study identified key regulatory elements (proteins/metabolites/bacteria) and pathways involved in the antitumor mechanisms of YQSJ, suggesting new potential therapeutic targets in CRC.

Deciphering core proteins of osteoporosis with iron accumulation by proteomics in human bone.

Iron accumulation is an independent risk factor for postmenopausal osteoporosis, but mechanistic studies of this phenomenon are still focusing on molecular and genetic researches in model animal. Osteoporosis with iron accumulation is a distinct endocrine disease with complicated pathogenesis regulated by several proteins. However, the comprehensive proteome-wide analysis of human bone is lacking. Using multiplex quantitative tandem mass tag-based proteomics, we detected 2900 and quantified 1150 proteins from bone of 10 postmenopausal patients undergoing hip replacement. Comparing with non-osteoporosis patients, a total of 75 differentially expressed proteins were identified, comprising 53 downregulated proteins and 22 upregulated proteins. These proteins primarily affect oxidoreductase activity, GTPase activity, GTP binding, and neural nucleus development, were mainly enriched in neural, angiogenesis and energy-related pathways, and formed complex regulatory networks with strong interconnections. We ultimately identified 4 core proteins (GSTP1, LAMP2, COPB1, RAB5B) that were significantly differentially expressed in the bone of osteoporosis patients with iron accumulation, and validated the changed protein level in the serum of the medical examination population. Our systemic analysis uncovers molecular insights for revealing underlying mechanism and clinical therapeutics in osteoporosis with iron accumulation.

Tandem mass tag-based proteomics analysis reveals the multitarget mechanisms of Phyllanthus emblica against liver fibrosis.

Phyllanthus emblica (PE), a traditional multiethnic herbal medicine, is commonly applied to treat liver diseases. Our previous study demonstrated that aqueous extract of PE (AEPE) could alleviate carbon tetrachloride (CCl4)-induced liver fibrosis in vivo, but the underlying molecular mechanisms are still unclear. The present study was undertaken to clarify the multitarget mechanisms of PE in treating liver fibrosis by proteomics clues. A CCl4-induced liver fibrosis rat model was established. The anti-liver fibrosis effects of chemical fractions from AEPE were evaluated by serum biochemical indicators and pathological staining. Additionally, tandem mass tag (TMT) - based quantitative proteomics technology was used to detect the hepatic differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, gene ontology (GO) enrichment and protein-protein interaction (PPI) network were used to perform bioinformatics analysis of DEPs. Western blot analysis was used to verify the key potential targets regulated by the effective fraction of AEPE. The low-molecular-weight fraction of AEPE (LWPE) was determined to be the optimal anti-liver fibrosis active fraction, that could significantly improve ALT, AST, HA, Col IV, PCIII, LN, Hyp levels and reduce the pathological fibrotic lesion of liver tissue in model rats. A total of 195 DEPs were screened after LWPE intervention. GO analysis showed that the DEPs were related mostly to extracellular matrix organization, actin binding, and extracellular exosomes. KEGG pathway analysis showed that DEPs are mainly related to ECM-receptor interactions, focal adhesion and PI3K-Akt signaling pathway. Combined with the GO, KEGG and Western blot results, COL1A2, ITGAV, TLR2, ACE, and PDGFRB may be potential targets for PE treatment of liver fibrosis. In conclusion, LWPE exerts therapeutic effects through multiple pathways and multiple targets regulation in the treatment of liver fibrosis. This study may provide proteomics clues for the continuation of research on liver fibrosis treatment with PE.

Tandem mass tag labeling to assess proteome differences between intermediate and very tender beef steaks.

Tenderness is considered as one of the most important quality attributes dictating consumers' overall satisfaction and future purchasing decisions of fresh beef. However, the ability to predict and manage tenderness has proven very challenging due to the numerous factors that contribute to variation in end-product tenderness. Proteomic profiling allows for global examination of differentially abundant proteins in the meat and can provide new insight into biological mechanisms related to meat tenderness. Hence, the objective of this study was to examine proteomic profiles of beef longissimus lumborum (LL) steaks varying in tenderness, with the intention to identify potential biomarkers related to tenderness. For this purpose, beef LL muscle samples were collected from 99 carcasses at 0 and 384 h postmortem. Based on Warner-Bratzler shear force values at 384 h, 16 samples with the highest (intermediate tender, IT) and lowest (very tender, VT) values were selected to be used for proteomic analysis in this study (n = 8 per category). Using tandem mass tag-based proteomics, a total of 876 proteins were identified, of which 51 proteins were differentially abundant (P < 0.05) between the tenderness categories and aging periods. The differentially identified proteins encompassed a wide array of biological processes related to muscle contraction, calcium signaling, metabolism, extracellular matrix organization, chaperone, and apoptosis. A greater (P < 0.05) relative abundance of proteins associated with carbohydrate metabolism and apoptosis, and a lower (P < 0.05) relative abundance of proteins involved in muscle contraction was observed in the VT steaks after aging compared with the IT steaks, suggesting that more proteolysis occurred in the VT steaks. This may be explained by the greater (P < 0.05) abundance of chaperonin and calcium-binding proteins in the IT steaks, which could have limited the extent of postmortem proteolysis in these steaks. In addition, a greater (P < 0.05) abundance of connective tissue proteins was also observed in the IT steaks, which likely contributed to the difference in tenderness due to added background toughness. The established proteomic database obtained in this study may provide a reference for future research regarding potential protein biomarkers that are associated with meat tenderness.

Tandem mass tag-based proteomics analysis of protein changes in the freezing and thawing cycles of Trachurus murphyi.

We investigated the proteome variations in Trachurus murphyi with different cycles of freezing and thawing (FT) under frozen storage. A total of 2,482 proteins were assessed quantitatively, of which 269 proteins were recognized as differential abundance proteins during the second FT cycle until the eighth FT cycle. Bioinformatics analysis on gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of Differential Analysis of Proteins (DAPs) indicated multiple DAPs engaged with the protein structure, metabolic enzymes, and protein turnover. In addition, some of the observed proteins were probably the underlying markers of protein oxidation (PO). The analysis of PO sites revealed the sites of PO, such as amino adipic semialdehydes, γ-glutamic semialdehydes, and Schiff bases. Bioinformatics analyses demonstrated the involvement of differentially expressed proteins in the Hippo signaling pathway (Ko04390), indicating strong protein degradation with greater numbers of FT cycles under frozen storage. It provides an insight into quality stability from a proteomics quality perspective at the molecular level. The results obtained have deepened our current understandings of the mechanisms that reveal variations in proteomes and quality, as well as help promote quality control of T. murphyi across the cold transportation chain. PRACTICAL APPLICATION: Temperature fluctuation is one of the core issues during frozen food storage and distribution faced by the frozen food industry. Fluctuation may result in microstructural changes, ice recrystallization, and protein change in frozen food products. Tandem mass tag-based methods were adopted to study proteome variations in Trachurus murphyi muscles under different cycles of freezing and thawing under frozen storage conditions in this paper. The results obtained have deepened our current understandings of the mechanisms that reveal variations in proteomes and quality, as well as help promote quality control of T. murphyi across the cold transportation chain.

The establishment of goat semen protein profile using a tandem mass tag-based proteomics approach

In this study, the complete proteome of goat ejaculated semen including spermatozoa and seminal plasma was established, applying a tandem mass tag (TMT) labeling together with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In seminal plasma, 2299 proteins were identified and 2098 proteins were quantified. The GO analysis demonstrated that 32% proteins were involved in metabolic activities. 46% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. Regarding molecular function, 40% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis indicated a primary involvement of the identified proteins in protein processing in endoplasmic reticulum, lysosome, and proteome. In spermatozoa, 2491 proteins were identified and quantified. 39% proteins are involved in metabolic activities. 48% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. 38% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis demonstrated their roles derived from the identified proteins in proteasome, glycolysis, pyruvate metabolism, and citrate cycle. Additionally, 1312 proteins were simultaneously presented in spermatozoa and seminal plasma. The involvement of 42% proteins in metabolic activities were observed. 47% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. The common proteins are mainly engaged on protein processing in endoplasmic reticulum, proteome, glycolysis, lysosome, and citrate cycle. Collectively, this study established the protein database of goat semen. More studies should be used to elucidate functionality of these identified proteins.

Tandem mass tag-based proteomics analysis reveals the effects of Guri Gumu-13 pill on drug-induced liver injury

The incidence of drug-induced liver injury (DILI) is second only to viral hepatitis and steatohepatitis in China, and DILI has become a serious public health problem that cannot be ignored. Guri Gumu-13 pill (GRGM) is a traditional Chinese medicine (TCM), which has a protective effect on liver diseases. However, the underlying therapeutic mechanisms of GRGM for DILI are still vague. In this study, the protective effect of GRGM on acetaminophen (APAP)-induced DILI was investigated based on the proteomics clues. The effects of GRGM on APAP-induced DILI in rats were studied using tandem mass tag (TMT)-based quantitative proteomics technology. Besides, western blotting was exerted to verify related proteins. Using the TMT-based quantitative proteomics approach, 237 proteins were identified as regulated in APAP-induced DILI and 58 proteins were regulated by GRGM. The 17 overlapping differentially expressed proteins (DEPs) were identified, and 7 proteins were inversely regulated. Enrichment analysis of KEGG indicated that metabolic pathways, linoleic acid metabolism, and retinol metabolism might be affected in DILI. Next, Cyp2c11, Aldh1a1, and Fads2 were verified with molecular biotechnology. GRGM exerts therapeutic effects through multi-pathways regulation in the treatment of DILI. This work may provide proteomics clues for the continuation of research on DILI treatment with GRGM.

Targeting kinome reprogramming in ESR1 fusion-driven metastatic breast cancer.

1085 Background: Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor-alpha positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain is replaced by C-terminal sequences from many different gene partners. Transcriptionally active ESR1 fusions promoted hormone-independent cell growth, motility and resistance to endocrine therapy. The diversity of partner genes creates a considerable diagnostic challenge and no targeted treatments exist for ESR1 translocated tumors. Thus, we have established a transcriptional signature to diagnose the presence of an active ESR1 fusion (PMID: 34711608) and developed novel targeted therapies against ESR1 fusion-driven biology. Methods: Fifteen ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines by lentiviral transduction. Cell growth was assayed by Alamar blue assay. A mass spectrometry (MS)-based Kinase Inhibitor Pulldown Assay (KIPA) and tandem mass tag-based proteomics were performed to identify ESR1 fusion-driven druggable kinases for subsequent pharmacological inhibition. Results: KIPA profiling demonstrated an increase of multiple receptor tyrosine kinases including RET in T47D cells expressing active ESR1 fusions. Inhibition of RET by repurposing an FDA-approved drug significantly suppressed ESR1 fusion-driven cell growth in vitro, suggesting that despite marked diversity in the 3’ partners, common kinase activities were elevated and targetable. Proteogenomic profiling, including whole exome sequencing, RNA sequencing, and MS-based proteomics and phosphoproteomics were further performed on 22 ER+ patient-derived xenograft (PDX) tumors, which demonstrated different degrees of estradiol dependence. These integrated “omic” profiles defined targetable genes/pathways and predict tumor subsets that could be responsive to kinase inhibition therapy from this biologically heterogeneous panel of PDX tumors. WHIM18, a PDX naturally harboring the ESR1-YAP1 fusion showed elevated level of RET and CDK4/6 pathways. The tumor volumes were significantly reduced by the RET inhibitor. CDK4/6 inhibitor treatment showed similar tumor reductions to RET inhibition. Interestingly, WHIM9 PDX that expressed wild-type ESR1 conferred a comparable kinome profile to WHIM18. The tumor growth was significantly suppressed by RET or CDK4/6 inhibition. Therefore, pharmacological experiments validated proteogenomics-predicted drug response in two tested ER+ PDX models. Conclusions: Proteogenomics characterization of PDX tumors can drive clinical trial hypotheses. Here, we reveal therapeutic kinase vulnerabilities in ESR1 fusion-driven tumors as exemplified by RET inhibition, which will lay the framework for future clinical trials.

Tandem Mass Tag-based proteomics analysis reveals the vital role of inflammation in traumatic brain injury in a mouse model

Proteomics is a powerful tool that can be used to elucidate the underlying mechanisms of diseases and identify new biomarkers. Therefore, it may also be helpful for understanding the detailed pathological mechanism of traumatic brain injury (TBI). In this study, we performed Tandem Mass Tag-based quantitative analysis of cortical proteome profiles in a mouse model of TBI. Our results showed that there were 302 differentially expressed proteins in TBI mice compared with normal mice 7 days after injury. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that these differentially expressed proteins were predominantly involved in inflammatory responses, including complement and coagulation cascades, as well as chemokine signaling pathways. Subsequent transcription factor analysis revealed that the inflammation-related transcription factors NF-κB1, RelA, IRF1, STAT1, and Spi1 play pivotal roles in the secondary injury that occurs after TBI, which further corroborates the functional enrichment for inflammatory factors. Our results suggest that inflammation-related proteins and inflammatory responses are promising targets for the treatment of TBI.

TMT-based proteomics analysis to screen potential biomarkers of Achyranthis Bidentatae Radix for osteoporosis in rats.

This study aimed to explore the possible mechanism of Achyranthis Bidentatae Radix for the treatment of osteoporosis using tandem mass tag-based proteomics technique combined with mass spectrometry. Proteomics techniques combined with bioinformatics were used to analyze the biological functions of differentially expressed proteins. In addition, western blotting was performed to verify the expression of related proteins. A total of 3,752 proteins were identifiable by proteomic analysis. Furthermore, 93 differentially expressed proteins were identified, of which 61 were upregulated and 32 were downregulated. Differentially expressed proteins were primarily associated with oxidative phosphorylation (p=4.8×10-4 ) pathways and involved in transmembrane transport (p=3.5×10-3 ), exocytic process (p=1.2×10-2 ), cellular developmental process (p=1.3×10-2 ), adenosine triphosphate metabolic process (p=1.0×10-2 ) and other biological processes. Western blotting analysis showed that MT-CYB and NDUFA9 were differentially expressed in the bone microenvironment of rats with osteoporosis. We speculated that they were potential biomarkers linked to osteoporosis. This study employed proteomics to explore the potential therapeutic targets of Achyranthis Bidentatae Radix to treat osteoporosis. This revealed that mitochondria are a new target for the treatment of glucocorticoid-induced osteoporosis.

In utero exposure to methylmercury impairs cognitive function in adult offspring: Insights from proteomic modulation

Methylmercury (MeHg) is a hazardous substance that has unique neurodevelopmental toxic effects. However, its molecular alteration profile, sensitive response biomarkers, and mechanism of neuronal injury remain largely unknown. Here, the effects of intrauterine methylmercury chloride (low-, medium- and high-dose groups: 0.6 mg/kg/d, 1.2 mg/kg/d, 2.4 mg/ kg /d, respectively) exposure on learning and memory were assessed in offspring rats by behavioral tests, pathological analysis and hippocampal proteomic analysis. The results suggested that intrauterine MeHg exposure impairs spatial learning and memory and leads a significant reduction in the number and dispersion scattered arrangement in the hippocampus of offspring. Furthermore, in the tandem mass tag-based proteomics analysis, compared with the control group, a total of 74 differentially expressed proteins (DEPs) were found in the MeHg exposure groups; specifically, 32 down-regulated and 42 up-regulated proteins were identified. In addition, the pathways enrichment analysis indicated that these DEPs are implicated in several biological processes, such as synaptic plasticity and energy metabolism, as well as various molecular functional categories. Simultaneously, MeHg reduced the postsynaptic density, diminished the active zone, amplified the synaptic cleft and changed the synaptic interface of pyramidal cells. Western blot analysis further revealed that MeHg significantly reduced the levels of Forkhead box protein (FOXP2), Synaptophysin (SYP) and Postsynaptic density protein 95 (PSD-95), and down-regulated the N-methyl-D-aspartate receptor 1 (NMDAR1), N-methyl-D-aspartate receptor 2 A (NR2A) and N-methyl-D-aspartate receptor 2B (NR2B). In general, from a functional perspective, most overlapping proteins were related to NMDA receptor-mediated glutamatergic signaling, which is an excitotoxicity mechanism known to influence learning and memory. These discoveries contribute to our understanding of the relationship between MeHg and cognitive deficits and provide insight into the protein mediators of this relationship and possible prospective early biomarkers.

Tandem mass tag-based proteomics analysis of type 2 diabetes mellitus with non-alcoholic fatty liver disease in mice treated with acupuncture.

Objective: To explore the proteomics profiles of hepatocytes of mice treated with acupuncture for type 2 diabetes mellitus (T2DM) with non-alcoholic fatty liver disease (NAFLD). Methods: We used a Tandem mass tag (TMT)-based quantitative proteomics approach to identify proteins with potential molecular mechanisms associated with acupuncture interventions for T2DM with NAFLD. Results: Acupuncture effectively improved body weight, blood glucose, and insulin levels in T2DM with NAFLD mouse models and reversed steatosis within hepatocytes. Quantitative TMT-based proteomics analysis identified a total of 4710 quantifiable proteins and 1226 differentially expressed proteins (DEPs) in the model control group (MCG) compared with the normal control group (NCG). The Acupuncture Treatment Group (ATG) presented in 122 DEPs was compared with the MCG group. We performed a bioinformatics analysis, which revealed that DEPs enriched in the KEGG pathway after acupuncture treatment were mainly involved in the PPAR signaling pathway, fatty acid biosynthesis, fatty acid metabolism, fatty acid elongation, fat digestion and absorption. We used parallel reaction monitoring (PRM) technology to explore the association of aldehyde oxidase 1 (Aox1), acyl-coenzyme A thioesterase 2 (Acot2), perilipin-2 (Plin2), acetyl-CoA carboxylase 1 (Acc), NADP-dependent malic enzyme (Me1), fatty acid synthase (Fasn), ATP-citrate synthase (Acly), fatty acid-binding protein, intestinal (Fabp2) with lipid synthesis, fatty acid oxidation, and hepatocyte steatosis. Conclusions: Our results show that acupuncture can regulate the protein expression of T2DM in the NAFLD mice model, and can effectively improve hepatocyte steatosis, and has potential benefits for the clinical treatment of this disease.

Tandem Mass Tag-Based Proteomics Reveals the effect of Electron Beam Irradiation on Metabolism-Related Differentially Expressed Proteins in Solenocera melantho Postmortem

ABSTRACT Postmortem metabolism in shrimp is an important factor affecting muscle quality during its preservation. Electron beam irradiation (EBI) can effectively kill microorganisms and inactivate enzymes. In this study, the tandem mass tag (TMT) method was used to study the changes in the muscle metabolism of Solenocera melantho postmortem before and after EBI (6 kGy). The TMT method identified a total of 656 proteins, including 120 differentially expressed proteins (DEPs) (56 upregulated, 64 downregulated). The analysis of these results showed that EBI can inactivate most enzymes and slow down their corresponding metabolic processes. These results also showed that irradiation can denature some proteins. The study revealed the effects of EBI on the muscle quality of S. melantho at the molecular level.