Mass Tag Research Articles

Proteomic analysis identified proteins that are differentially expressed in the flavonoid and carotenoid biosynthetic pathways of Camellia Nitidissima flowers

BackgroundCamellia nitidissima Chi is a popular ornamental plant because of its golden flowers, which contain flavonoids and carotenoids. To understand the regulatory mechanism of golden color formation, the metabolites of C. nitidissima petals at five different developmental stages were detected, a proteome map of petals was first constructed via tandem mass tag (TMT) analysis, and the accuracy of the sequencing data was validated via parallel reaction monitoring (PRM).ResultsNineteen color components were detected, and most of these components were carotenoids that gradually accumulated, while some metabolites were flavonoids that were gradually depleted. A total of 97,647 spectra were obtained, and 6,789 quantifiable proteins were identified. Then, 1,319 differentially expressed proteins (DEPs) were found, 55 of which belong to the flavonoid and carotenoid pathways, as revealed by pairwise comparisons of protein expression levels across the five developmental stages. Notably, most DEPs involved in the synthesis of flavonoids, such as phenylalanine ammonium lyase and 4-coumarate-CoA ligase, were downregulated during petal development, whereas DEPs involved in carotenoid synthesis, such as phytoene synthase, 1-deoxy-D-xylulose-5-phosphate synthase, and β-cyclase, tended to be upregulated. Furthermore, protein‒protein interaction (PPI) network analysis revealed that these 55 DEPs formed two distinct PPI networks closely tied to the flavonoid and carotenoid synthesis pathways. Phytoene synthase and chalcone synthase exhibited extensive interactions with numerous other proteins and displayed high connectivity within the PPI networks, suggesting their pivotal biological functions in flavonoid and carotenoid biosynthesis.ConclusionProteomic data on the flavonoid and carotenoid biosynthesis pathways were obtained, and the regulatory roles of the DEPs were analyzed, which provided a theoretical basis for further understanding the golden color formation mechanism of C. nitidissima.

Quantitative proteomics and multi-omics analysis identifies potential biomarkers and the underlying pathological molecular networks in Chinese patients with multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disorder caused by chronic inflammatory reactions in the central nervous system. Currently, little is known about the changes of plasma proteomic profiles in Chinese patients with MS (CpwMS) and its relationship with the altered profiles of multi-omics such as metabolomics and gut microbiome, as well as potential molecular networks that underlie the etiology of MS. To uncover the characteristics of proteomics landscape and potential multi-omics interaction networks in CpwMS, Plasma samples were collected from 22 CpwMS and 22 healthy controls (HCs) and analyzed using a Tandem Mass Tag (TMT)-based quantitative proteomics approach. Our results showed that the plasma proteomics pattern was significantly different in CpwMS compared to HCs. A total of 90 differentially expressed proteins (DEPs), such as LAMP1 and FCG2A, were identified in CpwMS plasma comparing to HCs. Furthermore, we also observed extensive and significant correlations between the altered proteomic profiles and the changes of metabolome, gut microbiome, as well as altered immunoinflammatory responses in MS-affected patients. For instance, the level of LAMP1 and ERN1 were significantly and positively correlated with the concentrations of metabolite L-glutamic acid and pro-inflammatory factor IL-17 (Padj < 0.05). However, they were negatively correlated with the amounts of other metabolites such as L-tyrosine and sphingosine 1-phosphate, as well as the concentrations of IL-8 and MIP-1α. This study outlined the underlying multi-omics integrated mechanisms that might regulate peripheral immunoinflammatory responses and MS progression. These findings are potentially helpful for developing new assisting diagnostic biomarker and therapeutic strategies for MS.

Proteomic profiling of extracellular vesicles in takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The mechanisms underlying its development and recovery are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and have been shown to be important in various diseases. Their involvement in cardiac conditions like TS is an emerging area of research. The primary objective of this study is to isolate and characterize EVs, as well as profile their proteomic profile, from the heart tissue of rats induced with TS. Methods Rats underwent TS induction through the infusion of 1 mg/kg isoprenaline over 15 minutes (TS24h), or were given saline (control). High-resolution echocardiography verified apical ballooning at 6 hours. After 24 hours, heart tissues from apical and basal segments were collected and processed. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, including a final iodixanol cushion separation for large and small EVs (Figure 1). Tandem mass tags and mass spectrometry were utilized for global proteomics and any differentially expressed proteins (DEPs) were identified. Analytical techniques such as volcano plots, heatmaps, enrichment analysis, and protein clustering/networks were employed. The strongest clusters (lowest False Discovery Rate and highest intensity) were selected, and their Gene Ontology (GO) terms/pathways were identified. Results Pure, cup-shaped, vesicles ranging from 50-800nm were successfully isolated (figure 1). EVs from apex of control and TS24h hearts had lower protein concentrations compared to their corresponding basal segments. Global proteomic analysis revealed a distinct protein profile in EVs from the apical segment of TS hearts at 24 hours. A total of 256 (TS24h-apex vs control-apex) and 561 proteins (TS24h-apex vs TS24h-base) were differentially expressed. No DEPs were observed when comparing the basal segments of TS24h and control hearts. Functional enrichment analysis of the DEPs showed an abundant change of biological processes related to metabolic lipid processes, inflammatory response, complement activation, reorganization of extracellular matrix. A downregulation was seen for biological processes related to mitochondrial ATP synthesis coupled electron transport and muscle contraction. Conclusion This study demonstrates a distinct EV protein profile in the apical segments of TS hearts, with marked changes in proteins related to metabolic lipid processes, inflammation, and mitochondrial activity. This extensive catalogue of novel proteins sets the stage for future research aimed at identifying potential therapeutic targets and diagnostic biomarkers, and enabling proof-of-concept studies in TS.

A Comprehensive Multi-Omics Study of Serum Alterations in Red Deer Infected by the Liver Fluke Fascioloides magna

Liver fluke infections are acknowledged as diseases with global prevalence and significant implications for both veterinary and public health. The large American liver fluke, Fascioloides magna, is a significant non-native parasite introduced to Europe, threatening the survival of local wildlife populations. The aim of this study was to analyze differences in the serum proteome and metabolome between F. magna-infected and control red deer. Serum samples from red deer were collected immediately following regular hunting operations, including 10 samples with confirmed F. magna infection and 10 samples from healthy red deer. A proteomics analysis of the serum samples was performed using a tandem mass tag (TMT)-based quantitative approach, and a metabolomics analysis of the serum was performed using an untargeted mass spectrometry-based metabolomics approach. A knowledge-driven approach was applied to integrate omics data. Our findings demonstrated that infection with liver fluke was associated with changes in amino acid metabolism, energy metabolism, lipid metabolism, inflammatory host response, and related biochemical pathways. This study offers a comprehensive overview of the serum proteome and metabolome in response to F. magna infection in red deer, unveiling new potential targets for future research. The identification of proteins, metabolites, and related biological pathways enhances our understanding of host–parasite interactions and may improve current tools for more effective liver fluke control.

Screening differentially expressed proteins to distinguish thymoma (B1 and B3) from thymic cysts based on tandem mass tag (TMT) technology

The therapeutic approach to thymic cysts remains a subject of controversy. Predicted biomarkers for identifying thymic cysts and thymoma (THYM) are crucial. In this research, patients diagnosed with thymic cysts (MTC, n = 6) and thymoma (B1, n = 6; B3, n = 6) were enrolled. Proteins of superior quality were subjected to TMT labeling and UPLC-MS, and differentially expressed proteins (DEPs) were identified. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interactive network analyses were applied to the DEPs. Some key differentially expressed genes(DEGs) were corroborated through GEPIA 32. The pan-cancer expression levels of key DEGs remarkably linked with prognosis were determined utilizing The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Eventually, 49 DEPs were identified in the B1 vs. MTC comparison (17 upregulated and 32 downregulated), 27 in the B3 vs. MTC comparison (8 upregulated and 19 downregulated), and 38 in the B3 vs. B1 comparison (9 upregulated and 29 downregulated). IL13RA1 (down), galectin-3 binding protein (LGALS3BP)(up), PRCSH (down), C3 (down), MXRA5 (down), TNN (down), CFHR1 (down), SUN3 (down) were jointly altered in both B1 vs. NZ and B3 vs. NZ. GEPIA validated that LGALS3BP was significantly upregulated in thymoma patients. In conclusion, LGALS3BP might be an essential biomarker to identify thymoma from the thymic cyst.

Comparative Analysis of Extracellular Vesicles from Cytotoxic CD8+ αβ T Cells and γδ T Cells.

Although belonging to different branches of the immune system, cytotoxic CD8+ αβ T cells and γδ T cells utilize common cytolytic effectors including FasL, granzymes, perforin and granulysin. The effector proteins are stored in different subsets of lysosome-related effector vesicles (LREVs) and released to the immunological synapse upon target cell encounter. Notably, in activated cells, LREVs and potentially other vesicles are continuously produced and released as extracellular vesicles (EVs). Presumably, EVs serve as mediators of intercellular communication in the local microenvironment or at distant sites. EVs of activated and expanded cytotoxic CD8+ αβ T cells or γδ T cells were enriched from culture supernatants by differential and ultracentrifugation and characterized by nanoparticle tracking analyses and Western blotting. For a comparative proteomic profiling, EV preparations from both cell types were isobaric labeled with tandem mass tags (TMT10plex) and subjected to mass spectrometry analysis. 686 proteins were quantified in EV preparations of cytotoxic CD8+ αβ T cells and γδ T cells. Both populations shared a major set of similarly abundant proteins, while much fewer proteins presented higher abundance levels in either CD8+ αβ T cells or γδ T cells. To our knowledge, we provide the first comparative analysis of EVs from cytotoxic CD8+ αβ T cells and γδ T cells.

4-Hydroxydictyolactone alleviates cerebral ischemia injury by regulating neuroinflammation and autophagy via AMPK signaling pathway

BackgroundCerebral ischemia (CI), a cerebrovascular disorder, is a major contributor to disability and mortality. Marine-derived compounds are an important source of new neuroprotective drug candidates. Xenicane-type diterpenes from brown algae of the genus Dictyota have exhibited potential neuroprotective effects against CI injury, attributed to their antioxidant properties. However, whether there are other underlying neuroprotective mechanisms of xenicane diterpenes against CI is still ambiguous. PurposeThis study aims to elucidate the neuroprotective efficacy and mechanism of 4-hydroxydictyolactone (HDTL) in the treatment of CI. MethodsThe LPS-induced BV2 cell model was used for anti-neuroinflammatory activity assay. Tandem Mass Tag (TMT)-based quantitative proteomics was employed to identify underlying mechanisms. The OGD/R-induced SH-SY5Y cell model and a MCAO mice model were used to assess the neuroprotective effect of HDTL against CI in vitro and in vivo. ResultsHDTL reduced inflammation in LPS-stimulated BV2 cells by inhibiting the IKK/IκB/NF-κB pathway and by enhancing AMPK phosphorylation. Additionally, in SH-SY5Y cells treated with OGD/R, HDTL facilitated autophagy and reduced apoptosis. The neuroprotective properties of HDTL were abrogated in AMPK- silenced SH-SY5Y cells. In MCAO mice, HDTL ameliorated CI injury as evidenced by decreases in neurological deficit scores and cerebral infarction. HDTL also promoted autophagy and reduced apoptosis in vivo through both the AMPK/mTOR and IKK/IκB/NF-κB pathways. ConclusionHDTL exhibits neuroprotective effects through regulating the AMPK/mTOR and IKK/IκB/NF-κB pathways. These findings suggest that HDTL is a promising therapeutic candidate for CI treatment.

KINOME-WIDE SYNTHETIC LETHAL SCREEN IDENTIfiES PANK4 AS A MODULATOR OF TEMOZOLOMIDE RESISTANCE IN GLIOBLASTOMA

Abstract AIMS Temozolomide (TMZ) represents the cornerstone of glioblastoma (GBM) therapy. However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterised. We aimed to uncover synthetic lethal partners of TMZ in GBM that could enhance the effect of TMZ, thus improving TMZ therapy response. METHOD A kinome-wide RNAi screen was performed in a TMZ-resistant GBM cell model. A panel of several TMZ-resistant and patient-derived GBM cell lines was implemented for in vitro validation of our findings through several phenotypic assays. In vivo TMZ-resistant GBM xenografts and immunohistochemical (IHC) analysis of IDH- wildtype GBM specimens were employed to assess the clinical relevance of our findings. Tandem Mass Tag (TMT)-based quantitative proteomic studies were undertaken to elucidate the underlying mechanisms. RESULTS Following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) was uncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborated the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. A Tandem Mass Tag (TMT)-based quantitative proteomic approach revealed that PANK4 abrogation leads to a significant downregulation of numerous proteins with central roles in cellular detoxification and cellular response to oxidative stress. Mechanistically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. CONCLUSION Our study provides novel insights into chemoresistance in GBM and unveils a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM.

TMT-based quantitative proteomic analysis of IBDV-infected CEF cells reveals a favorable role of chicken IRF10 in IBDV replication via suppressing type-I interferon expression

Infectious bursal disease (IBD) is an acute, highly contagious disease caused by infectious bursal disease virus (IBDV), causing huge economic losses to the poultry industry worldwide. Currently, the emerging variant strains of IBDV and new recombinants in the field are circulating in many countries and poses severe threats to the development of poultry industry. Elucidation of the pathogenesis of IBDV infection will be of great help to the development of vaccines for control of IBDV infection. In this study, liquid chromatography tandem-mass spectrometry (LC–MS/MS) combined with tandem mass tag (TMT) labeling was performed to determine the expressions of nucleus proteins in IBDV-infected chicken embryonic fibroblast (CEF) cells 24 h post-infection (hpi). Our data show that a total of 236 nucleus proteins were differentially expressed in IBDV-infected cells vs mock-infected controls, and that among those proteins, 171 were significantly upregulated while 65 downregulated. Bioinformatics analysis reveals that the differentially expressed proteins (DEPs) were mainly involved in immune response, DNA replication, mismatch repair, and RIG-I-like receptor (RLR) signaling. Consistently, the expression of ten selected upregulated genes (IRF10, IRF7, IRF1, STAT1, ATF3, GTF3A, CSRP3, RARB, BASP1, and NF-κB1) markedly increased as examined by quantitative real‐time PCR (qRT-PCR). Furthermore, the expression of IRF10 was upregulated both in the cytoplasm and nucleus of DF-1 cells as examined by Western Blot. Moreover, knockdown of IRF10 remarkably inhibited IBDV replication via promoting IFN-I response, and overexpression of IRF10 significantly suppressed type I interferon and ISGs expression in both mock and IBDV-infected cells, suggesting that IRF10 serve as a negative regulator for host antiviral response. These results provide clues to further investigation into host–IBDV interactions and the underlying mechanisms of IBDV infection.

SYNTAXIN OF PLANTS 132 underpins secretion of cargoes associated with salicylic acid signaling and pathogen defense.

Secretory trafficking in plant cells is facilitated by SNARE (soluble N-ethylamide-sensitive factor attachment protein receptor) proteins that drive membrane fusion of cargo-containing vesicles. In Arabidopsis, SYNTAXIN OF PLANTS 132 (SYP132) is an evolutionarily ancient SNARE that functions with syntaxins SYP121 and SYP122 at the plasma membrane. Whereas SYP121 and SYP122 mediate overlapping secretory pathways, albeit with differences in their importance in plant-environment interactions, the SNARE SYP132 is absolutely essential for plant development and survival. SYP132 promotes endocytic traffic of the plasma membrane H+-ATPase AHA1 and aquaporin PIP2;1, and it coordinates plant growth and bacterial pathogen immunity through PATHOGENESIS-RELATED1 (PR1) secretion. Yet, little else is known about SYP132 cargoes. Here, we used advanced quantitative Tandem Mass Tagging (TMT) mass spectrometry (MS) combined with immunoblot assays to track native secreted cargo proteins in the leaf apoplast. We found that SYP132 supports a basal level of secretion in Arabidopsis leaves, and its overexpression influences salicylic acid (SA) and jasmonic acid (JA) defence-related cargoes including PR1, PR2, and PR5 proteins. Impairing SYP132 function also suppressed defence-related secretory traffic when challenged with the bacterial pathogen Pseudomonas syringae. Thus, we conclude that, in addition to its role in hormone-related H+-ATPase cycling, SYP132 influences basal plant immunity.

Effects of 4.9 GHz Radiofrequency Field Exposure on Brain Metabolomic and Proteomic Characterization in Mice.

Electromagnetic exposure has become increasingly widespread, and its biological effects have received extensive attention. The purpose of this study was to explore changes in the metabolism profile of the brain and serum and to identify differentially expressed proteins in the brain after exposure to the 4.9 GHz radiofrequency (RF) field. C57BL/6 mice were randomly divided into a Sham group and an RF group, which were sham-exposed and continuously exposed to a 4.9 RF field for 35 d, 1 h/d, at an average power density (PD) of 50 W/m2. After exposure, untargeted metabolomics and Tandem Mass Tags (TMT) quantitative proteomics were performed. We found 104 and 153 up- and down-regulated differentially expressed metabolites (DEMs) in the RF_Brain group and RF_Serum group, and the DEMs were significantly enriched in glycerophospholipid metabolism. Moreover, 10 up-regulated and 51 down-regulated differentially expressed proteins (DEPs) were discovered in the RF group. Functional correlation analysis showed that most DEMs and DEPs showed a significant correlation. These results suggested that 4.9 GHz exposure induced disturbance of metabolism in the brain and serum, and caused deregulation of proteins in the brain.

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.

Bone and periosteum protein analysis via tandem mass tag quantitative proteomics in pediatric patients with osteomyelitis.

Bone healing is crucial in managing osteomyelitis after fracture fixation. Understanding the mechanism of extensive callus formation in pediatric osteomyelitis is highly important. This study aims to analyze bone and periosteum samples from pediatric patients to elucidate the essential processes involved in callus formation during osteomyelitis. The study included eight patients from our hospital: four with positive microbial culture who underwent osteomyelitis debridement and four who had osteotomy surgery as contral. We used tandem mass tag quantitative proteomics to investigate proteomic changes in bone and periosteum tissues obtained from these patients. Differential expression proteins were analyzed for their pathways through Gene Ontology (GO) annotation, GO enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction networks. A total of 4737 proteins were successfully identified. About 2224 differentially expressed proteins were detected in the bone tissues group and periosteum tissues group. Among the differentially expressed proteins, 10 protein genes in the bone group were associated with inflammation and osteogenesis, while in the periosteum group were nine. Cytochrome b-245, beta polypeptide (CYBB), nicotinamide phosphoribosyltransferase (NAMPT), tissue inhibitor of metalloproteinases 1 (TIMP-1), Raf-1 proto-oncogene, serine/threonine kinase (RAF-1), RELA proto-oncogene, NF-KB subunit (RELA), and sphingomyelin synthase 2 (SGMS2) may play an important role in callus formation in patients with osteomyelitis. This study provides novel clues for understanding callus formation in pediatric patients with osteomyelitis.

Heparin-enriched plasma proteome is significantly altered in Alzheimer’s disease

IntroductionHeparin binding proteins (HBPs) with roles in extracellular matrix assembly are strongly correlated to β-amyloid (Aβ) and tau pathology in Alzheimer’s disease (AD) brain and cerebrospinal fluid (CSF). However, it remains challenging to detect these proteins in plasma using standard mass spectrometry-based proteomic approaches.MethodsWe employed heparin-affinity chromatography, followed by off-line fractionation and tandem mass tag mass spectrometry (TMT-MS), to enrich HBPs from plasma obtained from AD (n = 62) and control (n = 47) samples. These profiles were then correlated to Aβ, tau and phosphorylated tau (pTau) CSF biomarkers and plasma pTau181 from the same individuals, as well as a consensus brain proteome network to assess the overlap with AD brain pathophysiology.ResultsHeparin enrichment from plasma was highly reproducible, enriched well-known HBPs like APOE and thrombin, and depleted high-abundant proteins such as albumin. A total of 2865 proteins, spanning 10 orders of magnitude in abundance, were measured across 109 samples. Compared to the consensus AD brain protein co-expression network, we observed that specific plasma proteins exhibited consistent direction of change in both brain and plasma, whereas others displayed divergent changes, highlighting the complex interplay between the two compartments. Elevated proteins in AD plasma, when compared to controls, included members of the matrisome module in brain that accumulate with Aβ deposits, such as SMOC1, SMOC2, SPON1, MDK, OLFML3, FRZB, GPNMB, and the APOE4 proteoform. Additionally, heparin-enriched proteins in plasma demonstrated significant correlations with conventional AD CSF biomarkers, including Aβ, total tau, pTau, and plasma pTau181. A panel of five plasma proteins classified AD from control individuals with an area under the curve (AUC) of 0.85. When combined with plasma pTau181, the panel significantly improved the classification performance of pTau181 alone, increasing the AUC from 0.93 to 0.98. This suggests that the heparin-enriched plasma proteome captures additional variance in cognitive dementia beyond what is explained by pTau181.ConclusionThese findings support the utility of a heparin-affinity approach coupled with TMT-MS for enriching amyloid-associated proteins, as well as a wide spectrum of plasma biomarkers that reflect pathological changes in the AD brain.

Mouse liver sinusoidal endothelial cell responses to the glucocorticoid receptor agonist dexamethasone.

Liver sinusoidal endothelial cells (LSECs) which make up the fenestrated wall of the hepatic sinusoids, are active scavenger cells involved in blood waste clearance and liver immune functions. Dexamethasone is a synthetic glucocorticoid commonly used in the clinic and as cell culture supplement. However, the response is dependent on tissue, cell type, and cell state. The aim of this study was to investigate the effect of dexamethasone on primary mouse LSECs (C57BL/6J); their viability (live-dead, LDH release, caspase 3/7 assays), morphology (scanning electron microscopy), release of inflammatory markers (ELISA), and scavenging functions (endocytosis assays), and associated biological processes and pathways. We have characterized and catalogued the proteome of LSECs cultured for 1, 10, or 48h to elucidate time-dependent and dexamethasone-specific cell responses. More than 6,000 protein IDs were quantified using tandem mass tag technology and advanced mass spectrometry (synchronous precursor selection multi-notch MS3). Enrichment analysis showed a culture-induced upregulation of stress and inflammatory markers, and a significant shift in cell metabolism already at 10h, with enhancement of glycolysis and concomitant repression of oxidative phosphorylation. At 48h, changes in metabolic pathways were more pronounced with dexamethasone compared to time-matched controls. Dexamethasone repressed the activation of inflammatory pathways (IFN-gamma response, TNF-alpha signaling via NF-kB, Cell adhesion molecules), and culture-induced release of interleukin-6, VCAM-1, and ICAM-1, and improved cell viability partly through inhibition of apoptosis. The mouse LSECs did not proliferate in culture. Dexamethasone treated cells showed upregulation of xanthine dehydrogenase/oxidase (Xdh), and the transcription regulator Foxo1. The drug further delayed but did not block the culture-induced loss of LSEC fenestration. The LSEC capacity for endocytosis was significantly reduced at 48h, independent of dexamethasone, which correlated with diminished expression of several scavenger receptors and C-type lectins and altered expression of proteins in the endocytic machinery. The glucocorticoid receptor (NR3C1) was suppressed by dexamethasone at 48h, suggesting limited effect of the drug in prolonged LSEC culture. Conclusion: The study presents a detailed overview of biological processes and pathways affected by dexamethasone in mouse LSECs in vitro.

TAOK2 Drives Opposing Cilia Length Deficits in 16p11.2 Deletion and Duplication Carriers.

Copy number variation (CNV) in the 16p11.2 (BP4-BP5) genomic locus is strongly associated with autism. Carriers of 16p11.2 deletion and duplication exhibit several common behavioral and social impairments, yet, show opposing brain structural changes and body mass index. To determine cellular mechanisms that might contribute to these opposing phenotypes, we performed quantitative tandem mass tag (TMT) proteomics on human dorsal forebrain neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSC) derived from 16p11.2 CNV carriers. Differentially phosphorylated proteins between unaffected individuals and 16p11.2 CNV carriers were significantly enriched for centrosomal and cilia proteins. Deletion patient-derived NPCs show increased primary cilium length compared to unaffected individuals, while stunted cilium growth was observed in 16p11.2 duplication NPCs. Through cellular shRNA and overexpression screens in human iPSC derived NPCs, we determined the contribution of genes within the 16p11.2 locus to cilium length. TAOK2, a serine threonine protein kinase, and PPP4C, a protein phosphatase, were found to regulate primary cilia length in a gene dosage-dependent manner. We found TAOK2 was localized at centrosomes and the base of the primary cilium, and NPCs differentiated from TAOK2 knockout iPSCs had longer cilia. In absence of TAOK2, there was increased pericentrin at the basal body, and aberrant accumulation of IFT88 at the ciliary distal tip. Further, pharmacological inhibition of TAO kinase activity led to increased ciliary length, indicating that TAOK2 negatively controls primary cilium length through its catalytic activity. These results implicate aberrant cilia length in the pathophysiology of 16p11.2 CNV, and establish the role of TAOK2 kinase as a regulator of primary cilium length.

DiDBiT-TMT: A Novel Method to Quantify Changes in the Proteomic Landscape Induced by Neural Plasticity.

Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.

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.

Proteomic Analysis of Crimped and Straight Wool in Chinese Tan Sheep.

Crimped wool in Tan sheep gradually transitions to straight wool after 35 days (the er-mao stage), which reduces its commercial value. To investigate the changes in wool proteins during this stage, we performed comparative proteomic analysis of the straight and crimped wool using tandem mass tag (TMT)-based quantification. The mean fur curvature (MFC) of crimped wool was significantly greater than that of straight wool (p < 0.001). We identified 1218 proteins between the two types of wool, including 50 keratins (Ks) and 10 keratin-associated proteins (KAPs). There were 213 differentially expressed proteins, including 13 Ks and 4 KAPs. Crimped wool showed relatively high abundances of KAP24-1, K84, K32, K82, and intermediate filament rod domain-containing protein (IRDC), whereas straight wool had relatively high abundances of K6A, K27, K80, KAP16-1, KAP27-1, and trichohyalin (TCHH). The expression levels of KAP16-1, KAP24-1, and KAP27-1 were related to the ratio of paracortex, which may be associated with wool crimp formation. Additionally, high expressions of TCHH, K27, and K6A in the inner root sheath (IRS) were linked to fiber fineness in straight wool. These findings provide insight into the overall expression and distribution patterns of Ks and KAPs, offering opportunities to improve wool quality and enhance its economic potential in the textile industry.

Salidroside rescues hypoxic cardiomyocytes by regulating the EGLN1/HIF‑1α pathway.

Myocardial infarction is characterized by oxygen deficiency caused by arterial flow restriction. Salidroside (SAL) protects against myocardial damage via antioxidant production and inhibition of apoptosis. The present study aimed to investigate potential rescue mechanism of SAL on hypoxic cardiomyocytes. H9C2 cardiomyocytes were divided into normoxia, hypoxia and hypoxia + SAL groups. The inhibitory rate of hypoxia and the optimal concentration and rescue effect of SAL were determined using Cell Counting Kit-8 assay and flow cytometry. Ca2+ concentration following hypoxia treatment and SAL intervention were detected by Fluo-4/acetoxymethyl. Tandem mass tag (TMT) proteomics was used to analyze the differential expression of hypoxia-associated proteins among the three groups. SAL exerted a protective effect on hypoxia-injured cardiomyocytes by enhancing aerobic metabolism during hypoxia and rescuing cardiomyocytes from hypoxic damage. SAL promoted cell proliferation, decreased apoptosis and increased Ca2+ levels in cell membranes of hypoxic cardiomyocytes. TMT proteomics results showed that the expression levels of intracellular hypoxia inducible factor-1 (HIF)-1α and Egl-9 family HIF 1 (EGLN1) in H9C2 cells were elevated under hypoxic conditions. However, SAL significantly decreased expression levels of HIF-1α and EGLN1. SAL inhibited mitochondrial calcium overload in hypoxic cardiomyocytes and attenuated expression of hypoxia-associated factors. SAL exerted its rescue effect on hypoxic cardiomyocytes through the EGLN1/HIF-1α pathway, thereby suppressing cardiomyocyte apoptosis, improving mitochondrial energy metabolism efficiency and rescuing cardiomyocytes from hypoxic injury.