Mass Tag-based Quantitative Proteomics Analysis Research Articles

Tandem mass tag-based quantitative proteomics analysis of modified atmosphere packaging in large yellow croaker (Pseudosciaena crocea) fillets during refrigerated storage

A tandem mass tagging-labeled proteomic approach was employed to explore the relationship between quality parameters and protein changes in large yellow croaker fillets refrigerated under carbon dioxide, oxygen, and nitrogen atmospheres. After 96 h, fillets stored in carbon dioxide and nitrogen showed improved texture, water-holding capacity, and color, compared to those stored in oxygen. Functional analysis respectively identified 117 and 65 differentially expressed proteins in carbon dioxide and nitrogen, including key proteins such as troponin, myosin light chain, actin, and collagen types IV and XIII, that were linked to extracellular adhesion, cytoskeleton integrity, energy metabolism, and membrane functions. Carbon dioxide and nitrogen were detrimental to the growth and reproduction of aerobic microorganisms. High concentrations of carbon dioxide can inhibit microbial activity, while nitrogen preserves freshness by regulating the pressure balance within the packaging. These findings provided a theoretical basis for the optimized gas-conditioned preservation of large yellow croaker fillets.

In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut.

The widespread sulfonamide resistance genes sul1, sul2, and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli, using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo. A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo. Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1, sul2, and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach.

Tandem mass tag-based quantitative proteomic analysis of metformin's inhibitory effects on ovarian cancer cells.

Metformin (MET), a type 2 diabetes treatment, has attracted increased attention for its potential antitumor properties; however, the precise mechanism underlying this activity remains unclear. Our previous in vivo and in vitro studies revealed MET's inhibitory effect on ovarian cancer, with the synergistic effects of MET and the MDM2 inhibitor RG7388 contributing to ovarian cancer treatment. This study further explores the mechanism underlying MET's inhibition of ovarian cancer. Following MET treatment, we analyzed the differentially expressed proteins in ovarian cancer cells using a tandem mass tag (TMT)-based proteomic approach coupled with bioinformatics. Using A2780 and SKOV3 ovarian cancer cells, we identified six upregulated and two downregulated proteins after MET treatment. Bioinformatics analysis revealed that these proteins predominately affect ovarian cancer cells by regulating iron ion transport, iron ion homeostasis, and mitochondrial and ribosomal functions. Validation via western blot confirmed MET-induced elevation of hydroxybutyrate dehydrogenase type 2 (BDH2) protein expression levels in A2780 and SKOV3 cells. Overall, our findings suggest that combining MET with other metabolic drugs, such as iron-chelating agents and mitochondrial inhibitors, may result in synergistic antitumor effects, thereby offering novel avenues for ovarian cancer treatment development.

Norgestrel causes oxidative damage to the digestive gland of the clam Mactra veneriformis

Increasingly, progestins are being detected in aquatic environments, especially in aquaculture areas, and concern about their adverse effects on aquatic organisms is growing. However, the mechanisms underlying progestin-induced oxidative stress and the antioxidant response of aquatic invertebrates to progestin exposure are unknown. Researchers recently reported that norgestrel (NGT) likely causes oxidative stress in the clam Mactra veneriformis. In this study, we assessed reactive oxygen species (ROS) levels, antioxidant enzyme activity, malonaldehyde content, ultrastructure, and DNA damage in the digestive gland of M. veneriformis exposed to 7.69 or 756.62 ng/L of NGT for 21 days. We also conducted tandem mass tag-based quantitative proteomic analysis to detect the protein response and gain insights into response mechanisms in clams exposed to NGT. We found that NGT enhanced oxidative phosphorylation via the wingless/integrated signaling pathway and triggered ROS generation. The alterations of antioxidant enzyme activities suggested that these enzymes are involved in scavenging NGT-induced ROS. NGT weakened antioxidant defense by suppressing the antioxidant synthesis-related AMP-activated protein kinase/forkhead box O and sirtuin 1/forkhead box O pathways. Additionally, cell membrane dissolution, mitochondrial cristae rupture, DNA damage, elevation of malonaldehyde content, and oxidation of lipid, protein, and glucose occurred due to compromised redox homeostasis. Our results highlight the oxidative stress, antioxidant response, and oxidative damage induced by NGT in M. veneriformis and shed light on ecological risks of progestins to aquatic invertebrates.

Tandem mass tag-based quantitative proteomics analysis of plasma and plasma exosomes in Parkinson’s disease

Tandem mass tag-based quantitative proteomics analysis of plasma and plasma exosomes in Parkinson’s disease

Electroacupuncture ameliorates cardiac dysfunction in myocardial ischemia model rats: a potential role of the hypothalamic-pituitary-adrenal axis.

To verify the hypothesis that electroacupuncture inhibits the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis regulating the expression of glial fibrillary acidic protein (GFAP) in the hippocampus of acute myocardial ischemia (AMI) rats. Sixty-six healthy male Sprague-Dawley rats were randomly divided into five groups: Sham, AMI (Model), electroacupuncture at Shenmen (HT7)-Tongli (HT5) segment (EA), non-acupoint electroacupuncture (Control), and Model + corticosterone (Model + CORT). AMI was induced occlusion of the left anterior descending coronary artery, followed by 3 d of electroacupuncture at Shenmen (HT7)-Tongli (HT5) segment. In the Control group, electroacupuncture was applied at points lying 5 and 10 mm from the base of the tail. The AMI + CORT group was injected with CORT (20 mg/kg) in saline. Hemorheology, electrocardiography (ECG), hematoxylin and eosin staining, and expression of glycogen phosphorylase BB (GPBB) and heart-type fatty acid-binding protein (H-FABP) were used to assess cardiac function. The effects of adrenocorticotropic hormone (ACTH) and CORT were evaluated by enzyme-linked immunosorbent assay. Protein expression in the Sham and Model groups were screened by tandem mass tag-based quantitative proteomics analysis. Protein expression was evaluated by Western blotting (vimentin and GFAP) and immunofluorescence staining (GFAP). Compared with the Sham group, the hemorheology indicators, heart rate, ECG-ST segment elevation, and GPBB and H-FABP levels were higher in Model rats. The EA group showed reductions in these indicators compared with the Model group. Similarly, in Model rats, the expression of ACTH and CORT were significantly increased compared with the Sham group. The EA group also showed reduced expression of ACTH and CORT. Importantly, proteomics analysis showed that vimentin was differentially expressed in Model rats. Compared with the Sham group, vimentin and GFAP expression in the hippocampus was increased in the Model group but decreased in the AMI + EA group. Additionally, intraperitoneal injection of CORT aggravated the expression of GPBB, H-FABP and GFAP. Our results suggested that electroacupuncture may protect against cardiac injury induced by AMI through regulation of HPA axis hyperactivity, and that hippocampal GFAP may play an important role in the regulation.

Proteomic analysis of acute radiation-induced rectal injury in rats

ObjectiveTo comprehensively elucidate overall protein alterations associated with acute radiation-induced rectal injury in rats. MethodsA rat model of acute radiation-induced rectal injury was established by irradiating rectal segments with a single dose of 17.5 ​Gy X-rays. These segments were then collected at 7 d and 10 ​d post-irradiation. Tandem mass tag-based quantitative proteomic analysis was then performed. Results65,526 peptides were identified, corresponding to 8,088 proteins. Hematoxylin-eosin staining revealed characteristic epithelial cell degeneration and necrosis, intestinal gland atrophy and dilatation, and interstitial inflammatory cell infiltration. Inflammation was more pronounced in the 10 d irradiation group than in the 7 d irradiation group. Overall, 127 up- and 108 downregulated proteins were identified at 7 d post-irradiation, and 122 up- and 44 downregulated proteins were identified at 10 d post-irradiation. Notably, 17 up- and 6 downregulated proteins were consistently co-expressed at both time points. The expression of three of these proteins was validated via real-time quantitative PCR: polypeptide YY (Pyy), thymidylate synthase (Tyms), and tetraspanin (CD9). Tyms transcript levels were significantly higher in irradiated rectal tissues (P ​< ​0.05). Pyy transcript levels were significantly higher at both time points (P ​< ​0.05). Finally, CD9 mRNA expression was significantly lower in both the 7 d and 10 d irradiation groups (P ​< ​0.05). ConclusionsThe potential targets were found to prevent and treat acute radiation-induced rectal injury in clinical practice.

Molecular and biochemical changes in Locusta migratoria (Orthoptera: Acrididae) infected with Paranosema locustae.

Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen's life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.

Fatty acid β-oxidation and mitochondrial fusion are involved in cardiac microvascular endothelial cell protection induced by glucagon receptor antagonism in diabetic mice.

The role of cardiac microvascular endothelial cells (CMECs) in diabetic cardiomyopathy is not fully understood. We aimed to investigate whether a glucagon receptor (GCGR) monoclonal antibody (mAb) ameliorated diabetic cardiomyopathy and clarify whether and how CMECs participated in the process. The db/db mice were treated with GCGR mAb or immunoglobulin G (as control) for 4 weeks. Echocardiography was performed to evaluate cardiac function. Immunofluorescent staining was used to determine microvascular density. The proteomic signature in isolated primary CMECs was analyzed by using tandem mass tag-based quantitative proteomic analysis. Some target proteins were verified by using western blot. Compared with db/m mice, cardiac microvascular density and left ventricular diastolic function were significantly reduced in db/db mice, and this reduction was attenuated by GCGR mAb treatment. A total of 199 differentially expressed proteins were upregulated in db/db mice versus db/m mice and downregulated in GCGR mAb-treated db/db mice versus db/db mice. The enrichment analysis demonstrated that fatty acid β-oxidation and mitochondrial fusion were the key pathways. The changes of the related proteins carnitine palmitoyltransferase 1B, optic atrophy type 1, and mitofusin-1 were further verified by using western blot. The levels of these three proteins were upregulated in db/db mice, whereas this upregulation was attenuated by GCGR mAb treatment. GCGR antagonism has a protective effect on CMECs and cardiac diastolic function in diabetic mice, and this beneficial effect may be mediated via inhibiting fatty acid β-oxidation and mitochondrial fusion in CMECs.

Uncovering pharmacological mechanisms of Phellinus linteus on focal segmental glomeruloscleosis rats through tandem mass tag-based quantitative proteomic analysis, network pharmacology analysis and experimental validation.

To explore the underlying molecular mechanism of (). We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-β1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-β1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-β1 signaling and may have potential applications in the treatment of FSGS.

Panax notoginseng saponin alleviates pulmonary fibrosis in rats by modulating the renin-angiotensin system

Ethnopharmacological relevancePulmonary fibrosis (PF) is a chronic, progressive, and often fatal interstitial lung disease. Traditional Chinese medicine formulations and their active ingredients have shown potential in the treatment of PF. Panax notoginseng saponin (PNS) is extracted from the widely used traditional Chinese medicinal herb Panax notoginseng (Burkill) F. H. Chen, exhibiting therapeutic effects in pulmonary diseases treatment. Aim of the studyThis study aimed to investigate the effects and elucidate possible potential mechanisms of PNS on bleomycin (BLM)-induced PF in rats. Materials and methodsPF was induced in rats by intratracheal administration of bleomycin (BLM, 5 mg/kg). After disease model induction, the rats were treated with PNS (50, 100, or 200 mg/kg per day) or pirfenidone (PFD, 50 mg/kg per day) for 28 days. Lung function, histopathological changes, collagen deposition, and E− and N-cadherin levels in lung tissue were evaluated. The mechanism of action of PNS was investigated using tandem mass tag-based quantitative proteomics analysis. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were performed to verify the proteomic results. ResultsPNS treatment improved lung function, ameliorated the BLM-induced increase in the lung coefficient, attenuated the degree of alveolar inflammation and fibrosis, and reduced the elevated collagen level in PF rats. PNS treatment also down-regulated the expression of N-cadherin while up-regulating the expression of E-cadherin. Proteomic and bioinformatic analyses revealed that the renin-angiotensin system (RAS) was closely related to the therapeutic effect of PNS. Immunohistochemistry, Western blot, and ELISA results indicated that PNS exerted its anti-fibrotic effect via regulation of the balance between the angiotensin-converting enzyme (ACE)-angiotensin (Ang)II-AngII receptor type 1 (AT1R) and ACE2-Ang(1–7)-MasR axes. ConclusionsPNS ameliorates BLM-induced PF in rats by modulating the RAS homeostasis, and is a new potential therapeutic agent for PF.

Tandem mass tag-based quantitative proteomic analysis identification of succinylation related proteins in pathogenesis of thoracic aortic aneurysm and aortic dissection.

Thoracic aortic aneurysm and dissection (TAAD) are devastating cardiovascular diseases with a high rate of disability and mortality. Lysine succinylation, a newly found post-translational modification, has been reported to play an important role in cardiovascular diseases. However, how succinylation modification influences TAAD remains obscure. Ascending aortic tissues were obtained from patients with thoracic aortic aneurysm (TAA, n = 6), thoracic aortic dissection (TAD) with pre-existing aortic aneurysm (n = 6), and healthy subjects (n = 6). Global lysine succinylation level was analyzed by Western blotting. The differentially expressed proteins (DEPs) were analyzed by tandem mass tag (TMT) labeling and mass spectrometry. Succinylation-related proteins selected from the literature review and AmiGO database were set as a reference inventory for further analysis. Then, the pathological aortic sections were chosen to verify the proteomic results by Western blotting and qRT-PCR. The level of global lysine succinylation significantly increased in TAA and TAD patients compared with healthy subjects. Of all proteins identified by proteomic analysis, 197 common DEPs were screened both in TAA and TAD group compared with the control group, of which 93 proteins were significantly upregulated while 104 were downregulated. Among these 197 DEPs, OXCT1 overlapped with the succinylation-related proteins and was selected as the target protein involved in thoracic aortic pathogenesis. OXCT1 was further verified by Western blotting and qRT-PCR, and the results showed that OXCT1 in TAA and TAD patients was significantly lower than that in healthy donors (p < 0.001), which was consistent with the proteomic results. OXCT1 represents novel biomarkers for lysine succinylation of TAAD and might be a therapeutic target in the future.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid β-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

Pivotal biological processes and proteins for selenite reduction and methylation in Ganoderma lucidum.

Microbial transformations, especially the reduction and methylation of Se oxyanion, have gained significance in recent years as effective detoxification methods. Ganoderma lucidum is a typical Se enrichment resource that can reduce selenite to elemental Se and volatile Se metabolites under high selenite conditions. However, the detailed biological processes and reduction mechanisms are unclear. In this study, G. lucidum reduced selenite to elemental Se and further aggregated it into Se nanoparticles with a diameter of <200nm, simultaneously accompanied by the production of pungent, odorous, and volatile methyl-selenium metabolites. Tandem mass tag-based quantitative proteomic analysis revealed thioredoxin 1, thioredoxin reductase (NADPH), glutathione reductase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and cystathionine gamma-lyase as proteins involved in selenite reduction and methylation. Furthermore, the high expression of proteins associated with cell structures that prompted cell lysis may have facilitated Se release. The upregulation of proteins involved in the defense reactions was also detected, reflecting their roles in the self-defense mechanism. This study provides novel insights into the vital role of G. lucidum in mediating Se transformation in the biogeochemical Se cycle and contributes to the application of fungi in Se bioremediation.

Comparative Tandem Mass Tag-Based Quantitative Proteomics Analysis of Liver Against Chronic Hypoxia: Molecular Insights Into Metabolism in Rats.

Xu, Jin, Shenhan Gao, Mingyuan Xin, Wenjie Chen, Kaikun Wang, Wenjing Liu, Xinzong Yan, Sinan Peng, and Yanming Ren. Comparative tandem mass tag-based quantitative proteomics analysis of liver against chronic hypoxia: molecular insights into metabolism in rats. High Alt Med Biol. 24:49-58, 2023. Objective: Using a metabolomic approach, we uncovered key regulators in metabolism from tandem mass tag (TMT)-based proteomic analysis in animals chronically exposed to hypoxia. Methods: Sixteen Sprague-Dawley rats (n = 8 per group) were exposed to chronic normoxia or hypoxia (380 mmHg corresponding to a simulated altitude of 5,500 m) for 35 consecutive days. Hypoxia-induced alterations in metabolic pathways were analyzed from TMT-based proteomic analysis, complemented by western blot validation of key regulators. Results: We profiled biochemical parameters and serum lipids, found that serum alanine aminotransferase and blood glucose were not significantly changed due to chronic hypoxia. However, serum triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein (LDL) were significantly affected by chronic hypoxia. And the levels of LDL nearly doubled (p < 0.05) after hypoxia exposure for 35 days. Through Kyoto Encyclopedia of Genes and Genomes classification, we found several metabolic pathways were enriched, including lipid metabolism, cofactor and vitamin metabolism, amino acids metabolism, carbohydrate metabolism, and energy metabolism. To explore the potential functions of proteins in metabolic pathways that become a coordinated shift under chronic hypoxic conditions, Gene Ontology and pathway analysis were carried out on differentially expressed proteins. As the co-expression network shown in Figure, we identified the most significant differentially expressed proteins after chronic hypoxic changes in the livers of rats. Furthermore, we validated the gene expression profiles at the protein level using western blot. Results of western blot were in accordance with our quantitative polymerase chain reaction findings. The levels of fatty acid synthase and aquaporin 1 were significantly downregulated after 35 days and the levels of ATP citrate lyase, 2'-5'-oligoadenylate synthetase 1A, aldehyde dehydrogenase 2, and Ras-related protein Rap-1A were significantly upregulated after 35 days. Conclusions: Although this study cannot completely account for all the molecular mechanisms in rats, we provide a good analysis of protein expression and profiling of rats under chronic hypoxia conditions.

Tandem mass tag-based quantitative proteomics analysis reveals the new regulatory mechanism of progranulin in influenza virus infection.

Progranulin (PGRN) plays an important role in influenza virus infection. To gain insight into the potential molecular mechanisms by which PGRN regulates influenza viral replication, proteomic analyzes of whole mouse lung tissue from wild-type (WT) versus (vs) PGRN knockout (KO) mice were performed to identify proteins regulated by the absence vs. presence of PGRN. Our results revealed that PGRN regulated the differential expression of ALOX15, CD14, CD5L, and FCER1g, etc., and also affected the lysosomal activity in influenza virus infection. Collectively these findings provide a panoramic view of proteomic changes resulting from loss of PGRN and thereby shedding light on the functions of PGRN in influenza virus infection.

Tandem mass tag-based quantitative proteomic analysis of effects of multiple sevoflurane exposures on the cerebral cortex of neonatal and adult mice.

Sevoflurane is the most commonly used general anesthetic in pediatric surgery, but it has the potential to be neurotoxic. Previous research found that long-term or multiple sevoflurane exposures could cause cognitive deficits in newborn mice but not adult mice, whereas short-term or single inhalations had little effect on cognitive function at both ages. The mechanisms behind these effects, however, are unclear. In the current study, 6- and 60-day-old C57bl mice in the sevoflurane groups were given 3% sevoflurane plus 60% oxygen for three consecutive days, each lasting 2 hours, while those in the control group only got 60% oxygen. The cortex tissues were harvested on the 8th or 62nd day. The tandem mass tags (TMT)pro-based quantitative proteomics combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, Golgi staining, and western blotting analysis were applied to analyze the influences of multiple sevoflurane anesthesia on the cerebral cortex in mice with various ages. The Morris water maze (MWM) test was performed from postnatal day (P)30 to P36 or P84 to P90 after control or multiple sevoflurane treatment. Sevoflurane anesthesia affected spatial learning and memory and diminished dendritic spines primarily in newborn mice, whereas mature animals exhibited no significant alterations. A total of 6247 proteins were measured using the combined quantitative proteomics methods of TMTpro-labeled and LC-MS/MS, 443 of which were associated to the age-dependent neurotoxic mechanism of repeated sevoflurane anesthesia. Furthermore, western blotting research revealed that sevoflurane-induced brain damage in newborn mice may be mediated by increasing the levels of protein expression of CHGB, PTEN, MAP2c, or decreasing the level of SOD2 protein expression. Our findings would help to further the mechanistic study of age-dependent anesthetic neurotoxicity and contribute to seek for effective protection in the developing brain under general anesthesia.

Proteomic analysis reveals allelopathic responsive mechanisms for para-hydroxybenzoic acid in poplars

The allelochemical para-hydroxybenzoic acid (pHBA) is a serious threat to poplars subjected to continuous cropping. In order to elucidate the responsive mechanisms of plants to pHBA stress, we here performed tandem mass tag-based quantitative proteomic analysis combined with physiological, transcriptional, and metabolic analyses by using H2O2 treatment as a positive control. H2O2 and O2•− levels and the activities of several antioxidant enzymes increased under pHBA stress. In total, 289 differentially expressed proteins were identified in the 84K poplar leaves under pHBA stress; among them, 161 and 128 proteins were upregulated and downregulated, respectively. The pHBA-responsive protein analysis showed that the antioxidant system, signal transduction, carbohydrate metabolism, biosynthesis of secondary metabolites, gene expression regulation, and many other biological processes were altered in poplars to cope with this stress. Overall, this study provides a new insight for the molecular mechanisms underlying pHBA stress in plants and may have potential application values in poplar genetic breeding for overcoming continuous cropping obstacles.

Tandem Mass Tag-Based Quantitative Proteomics Analysis of Gonads Reveals New Insight into Sexual Reversal Mechanism in Chinese Soft-Shelled Turtles.

Simple SummaryThe Chinese soft-shelled turtle has obvious sex dimorphism, which is an important aquatic economic species in China. Exogenous hormones can cause the sexual reversal of P. sinensis, but the molecular mechanism remains unclear. Here, TMT-based quantitative proteomics analysis of four types of P. sinensis (i.e., female (F), male (M), pseudo-female (PF), pseudo-male (PM)) gonads were performed. We found that there were common pathways, such as focal adhesion, endocytosis, apoptosis, ribosome, and spliceosome, which both played crucial roles in two comparison groups, including F vs. PM and M vs. PF. Furthermore, these differentially expressed proteins were associated with various biological processes, such as embryonic development and catabolic process, which were closely related to the sexual reversal of P. sinensis.Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis.

NUPR1 promotes the proliferation and metastasis of oral squamous cell carcinoma cells by activating TFE3-dependent autophagy

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy, and metastasis accounts for the poor prognosis of OSCC. Autophagy is considered to facilitate OSCC development by mitigating various cellular stresses; nevertheless, the mechanisms of autophagy in OSCC cell proliferation and metastasis remain unknown. In our study, high-sensitivity label-free quantitative proteomics analysis revealed nuclear protein 1 (NUPR1) as the most significantly upregulated protein in formalin-fixed paraffin-embedded tumour samples derived from OSCC patients with or without lymphatic metastasis. Moreover, NUPR1 is aberrantly expressed in the OSCC tissues and predicts low overall survival rates for OSCC patients. Notably, based on tandem mass tag-based quantitative proteomic analysis between stable NUPR1 knockdown OSCC cells and scrambled control OSCC cells, we confirmed that NUPR1 maintained autophagic flux and lysosomal functions by directly increasing transcription factor E3 (TFE3) activity, which promoted OSCC cell proliferation and metastasis in vitro and in vivo. Collectively, our data revealed that the NUPR1–TFE3 axis is a critical regulator of the autophagic machinery in OSCC progression, and this study may provide a potential therapeutic target for the treatment of OSCC.