Mass Tag Research Articles

In-Depth Proteome Profiling of the Hippocampus of LDLR Knockout Mice Reveals Alternation in Synaptic Signaling Pathway.

The low-density lipoprotein receptor (LDLR) is a major apolipoprotein receptor that regulates cholesterol homeostasis. LDLR deficiency is associated with cognitive impairment by the induction of synaptopathy in the hippocampus. Despite the close relationship between LDLR and neurodegenerative disorders, proteomics research for protein profiling in the LDLR knockout (KO) model remains insufficient. Therefore, understanding LDLR KO-mediated differential protein expression within the hippocampus is crucial for elucidating a role of LDLR in neurodegenerative disorders. In this study, we conducted first-time proteomic profiling of hippocampus tissue from LDLR KO mice using tandem mass tag (TMT)-based MS analysis. LDLR deficiency induces changes in proteins associated with the transport of diverse molecules, and activity of kinase and catalyst within the hippocampus. Additionally, significant alterations in the expression of components in the major synaptic pathways were found. Furthermore, these synaptic effects were verified using a data-independent acquisition (DIA)-based proteomic method. Our data will serve as a valuable resource for further studies to discover the molecular function of LDLR in neurodegenerative disorders.

Ultra-inert lanthanide chelates as mass tags for multiplexed bioanalysis.

Coordination compounds of lanthanides are indispensable in biomedical applications as MRI contrast agents and radiotherapeutics. However, since the introduction of the chelator DOTA four decades ago, there has been only limited progress on improving their thermodynamic stability and kinetic inertness, which are essential for safe in vivo use. Here, we present ClickZip, an innovative synthetic strategy employing a coordination-templated formation of a 1,5-triazole bridge that improves kinetic inertness up to a million-fold relative to DOTA, expanding utility of lanthanide chelates beyond traditional uses. Acting as unique mass tags, the ClickZip chelates can be released from (biological) samples by acidic hydrolysis, chromatographically distinguished from interfering lanthanide species, and sensitively detected by mass spectrometry. Lanthanides enclosed in ClickZip chelates are chemically almost indistinguishable, providing a more versatile alternative to chemically identical isotopic labels for multiplexed analysis. The bioanalytical potential is demonstrated on tagged cell-penetrating peptides in vitro, and anti-obesity prolactin-releasing peptides in vivo.

Comparative transcriptomic and proteomic analyses of two salt-tolerant alfalfa (Medicago sativa L.) genotypes: investigation of the mechanisms underlying tolerance to salt

Abiotic stressors such as salt stress restrict plant development and output, which lowers agricultural profitability. In this study, alfalfa (Medicago sativa L.) varieties with different levels of salt tolerance were examined using high-throughput RNA sequencing (RNA-Seq) and Tandem Mass Tags (TMT) technologies to study the reactions of the root systems to salt stress, from transcriptomics and proteomics perspectives. The varieties Atlantic (AT) and Zhongmu-1 (ZM-1) were selected and evaluated after 2 h and 6 h of treatment with 150 mM NaCl. The results showed that under salt stress for 2 h, 1810 differentially expressed genes (DEGs) and 160 differentially expressed proteins (DEPs) in AT were screened, while 9341 DEGs and 193 DEPs were screened in ZM-1. Under salt stress for 6 h, 7536 DEGs and 118 DEPs were screened in AT, while 11,754 DEGs and 190 DEPs were screened in ZM-1. Functional annotation and pathway enrichment analyses indicated that the DEGS and DEPs were mainly involved in the glutathione metabolism, biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, carbon fixation in photosynthetic organisms, and photosynthesis pathways. A series of genes related to salt tolerance were also identified, including GSTL3 and GSTU3 of the GST gene family, PER5 and PER10, of the PER gene family, and proteins such as APR and COMT, which are involved in biosynthesis of secondary metabolites. This study provides insights into salt resistance mechanisms in plants, and the related genes and metabolic pathways identified may be helpful for alfalfa breeding in the future.

The biochemical effects of carotenoids in orange carrots on the colonic proteome in a mouse model of diet-induced obesity

IntroductionCarotenoids are naturally occurring pigments in plants and are responsible for the orange, yellow, and red color of fruits and vegetables. Carrots are one of the primary dietary sources of carotenoids. The biological activities of carotenoids in higher organisms, including their immunomodulatory activities, are well documented in most tissues but not the large intestine. The gastrointestinal barrier acts as a line of defense against the systemic invasion of pathogenic bacteria, especially at the colonic level.MethodsTo test whether carotenoids in orange carrots can alleviate obesity-associated gut inflammation and strengthen the intestinal barrier function, male C57BL/6J mice were randomized to one of four experimental diets for 20 weeks (n = 20 animals/group): Low-fat diet (LFD, 10% calories from fat), high-fat diet (HFD, 45% calories from fat), HFD with white carrot powder (HFD+WC), or HFD with orange carrot powder (HFD + OC). Colon tissues were harvested to analyze the biochemical effects of carotenoids in carrots. The distal sections were subjected to isobaric labeling-based quantitative proteomics in which tryptic peptides were labeled with tandem mass tags, followed by fractionation and LC-MS/MS analysis in an Orbitrap Eclipse Tribrid instrument.ResultsHigh-performance liquid chromatography results revealed that the HFD+WC pellets were carotenoid-deficient, and the HFD+OC pellets contained high concentrations of provitamin A carotenoids, specifically α-carotene and β-carotene. As a result of the quantitative proteomics, a total of 4410 differentially expressed proteins were identified. Intestinal barrier-associated proteins were highly upregulated in the HFD+OC group, particularly mucin-2 (MUC-2). Upon closer investigation into mucosal activity, other proteins related to MUC-2 functionality and tight junction management were upregulated by the HFD+OC dietary intervention.DiscussionCollectively, our findings suggest that carotenoid-rich foods can prevent high-fat diet-induced intestinal barrier disruption by promoting colonic mucus synthesis and secretion in mammalian organisms. Data are available via ProteomeXchange with identifier PXD054150.

Metabolomics and proteomics insights into hepatic responses of weaned piglets to dietary Spirulina inclusion and lysozyme supplementation

BackgroundStudying the effect of dietary Spirulina and lysozyme supplementation on the metabolome and proteome of liver tissue contributes to understanding potential hepatic adaptations of piglets to these novel diets. This study aimed to understand the influence of including 10% Spirulina on the metabolome and proteome of piglet liver tissue. Three groups of 10 post-weaned piglets, housed in pairs, were fed for 28 days with one of three experimental diets: a cereal and soybean meal-based diet (Control), a base diet with 10% Spirulina (SP), and an SP diet supplemented with 0.01% lysozyme (SP + L). At the end of the trial, animals were sacrificed and liver tissue was collected. Metabolomics analysis (n = 10) was performed using NMR data analysed with PCA and PLS-DA. Proteomics analysis (n = 5) was conducted using a filter aided sample preparation (FASP) protocol and Tandem Mass Tag (TMT)-based quantitative approach with an Orbitrap mass spectrometer.ResultsGrowth performance showed an average daily gain reduction of 9.5% and a feed conversion ratio increase of 10.6% in groups fed Spirulina compared to the control group. Metabolomic analysis revealed no significant differences among the groups and identified 60 metabolites in the liver tissue. Proteomics analysis identified 2,560 proteins, with 132, 11, and 52 differentially expressed in the Control vs. SP, Control vs. SP + L and SP vs. SP + L comparisons, respectively. This study demonstrated that Spirulina enhances liver energy conversion efficiency, detoxification and cellular secretion. It improves hepatic metabolic efficiency through alterations in fatty acid oxidation (e.g., upregulation of enzymes like fatty acid synthase and increased acetyl-CoA levels), carbohydrate catabolism (e.g., increased glucose and glucose-6-phosphate), pyruvate metabolism (e.g., higher levels of pyruvate and phosphoenolpyruvate carboxykinase), and cellular defence mechanisms (e.g., upregulation of glutathione and metallothionein). Lysozyme supplementation mitigates some adverse effects of Spirulina, bringing physiological responses closer to control levels. This includes fewer differentially expressed proteins and improved dry matter, organic matter and energy digestibility. Lysozyme also enhances coenzyme availability, skeletal myofibril assembly, actin-mediated cell contraction, tissue regeneration and development through mesenchymal migration and nucleic acid synthesis pathways.ConclusionsWhile Spirulina inclusion had some adverse effects on growth performance, it also enhanced hepatic metabolic efficiency by improving fatty acid oxidation, carbohydrate catabolism and cellular defence mechanisms. The addition of lysozyme further improved these benefits by reducing some of the negative impacts on growth and enhancing nutrient digestibility, tissue regeneration, and overall metabolic balance. Together, Spirulina and lysozyme demonstrate potential as functional dietary components, but further optimization is needed to fully realize their benefits without compromising growth performance.

Aptamer-functionalized magnetic blade spray coupled with nucleic acid dye-based mass tag strategy for miniature mass spectrometry analysis of endoglin

Ambient ionization mass spectrometry (AIMS) allows rapid analysis of targets, while its overall selectivity is somewhat limited due to the lack of chromatographic separation. Recently, magnetic blade spray (MBS) has enhanced AIMS by incorporating immunomagnetic beads instead of the traditional coated blade spray (CBS) coating, thereby improving selectivity and sensitivity by targeted analyte detection and reducing background interference. In this study, an aptamer-functionalized and nucleic acid dye (GelRed)-loaded MS probe (AGMP) was developed and employed with MBS-based miniature mass spectrometer. Specifically, AGMP was assembled using aptamer-functionalized magnetic nanoparticles loaded with GelRed as mass tags for highly sensitive analysis of endoglin (CD105). For preparation of AGMP, the CD105 binding aptamer of End-A2 was first selected through three rounds of capillary electrophoresis (CE)-SELEX with an optimal affinity of 62.3 pM. After optimizing the critical parameters that affected adsorption, desorption, and ionization efficiency, this method displayed satisfactory sensitivity with detection and quantitation limits of 0.2 and 1 ng/mL, respectively, as well as reliable recoveries of 90.1–106.8% with relative standard deviations of 1.6–5.4%. Besides, the method effectively mitigated the matrix effects with a slope deviation of 10.03%, and exhibited good selectivity and environmental friendliness. Furthermore, this AGMP-based MBS strategy was successfully applied for CD105 detection in serum samples, demonstrating its potential for sensitive and on-site biomolecule analysis in complex matrices.

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.

Mass-tagged self-assembled nanoprobe reveals the transport of PD-L1 from cancer cells to tumor-educated platelets

BackgroundThe expression level of immune checkpoint proteins detected by tissue biopsy is currently used as a predictive biomarker for immune checkpoint blockade (ICB) therapy. However, tissue biopsy is susceptible to invasive sample collection procedures, significant sampling heterogeneity, and the difficulty of repeated sampling. Therefore, liquid biopsy of blood samples is becoming an alternative choice for immune checkpoint protein detection. Among various vesicles in blood, platelets can obtain cancer information to form a specific group called tumor-educated platelets (TEPs). The platelet-derived proteins in TEPs may have a predictive potential in ICB therapy. ResultsIn this study, a photo-cleavable mass-tagged self-assembled (SAMT) nanoprobe with signal amplification was developed for the quantitative detection of PD-L1. The SAMT probe was assembled by photo-cleavable mass tags, PD-L1 aptamer, and amphiphilic polymer. After binding with PD-L1 on the platelet, the probe can release mass tags with UV light exposure. The amount of the mass tag, representing that of PD-L1, was subsequently determined by mass spectrometry. The assay sensitivity can be greatly improved by up to four orders of magnitude, achieving a detection limit of 10 fM. This assay was subsequently applied to cancer cells and platelet samples from non-small cell lung cancer (NSCLC) patients. The patients with higher tumor stages, higher degrees of lymph node invasion, and better ICB response had higher PD-L1 levels on platelets. Further investigation revealed that PD-L1 on the platelets was transported from cancer cells, providing evidence for the existence of TEPs. SignificanceThe SAMT probe can amplify the signal of the target molecule into that of multiple mass tags, achieving ultrasensitive ICB protein quantitative detection in platelets. Moreover, the employed SAMT assay not only revealed PD-L1 transport from cancer cells to platelets but also confirmed the presence of TEPs.

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.