Mass Tag Research Articles

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Proteomic characterization of small vessel pathology in PSEN1 E280A familial Alzheimer’s disease

AbstractBackgroundVascular pathology is a main contributor to Alzheimer’s disease (AD) progression and severity. We have previously characterized vascular pathology in the PSEN1E280A Colombian kindred. In postmortem analyses we identified that the occipital cortex (OC) is the most affected region by small vessel disease (SVD), including other cortical areas and basal ganglia. Interestingly, frontal cortex (FC) is spared from tau pathology in protected PSEN1E280A cases, while the OC is not. Here we study the proteome of isolated vessels from frontal and occipital cortices to identify differences in molecular pathways.MethodApproximately 400 mg of FC and OC of postmortem brain tissue from PSEN1E280A familial AD (FAD, n = 10), sporadic AD (SAD, n = 10) and controls (HC, n = 5) were homogenized, centrifuged at 2000 g / 5 minutes before using gradient centrifugation at 10000 g / 15 minutes with dextran to receive the microvessel containing pellet which was retrieved and resuspended in lysis buffer for tandem mass tag (TMT) proteomics. Proteomics spectral data were matched to peptide sequences in the human UniProt protein database using the MaxQuant software package. The MaxQuant output files were analysed by MSstatsTMT using R software for statistical analysis. clusterProfiler package was used to perform Gene Ontology enrichment analyses.ResultMicrovessels isolated from AD OC showed 1597 significantly dysregulated proteins when compared to controls, while FC showed 523. FAD showed the majority of upregulated proteins in FC (181 > 88 in SAD), and OC (428 > 84). This pattern was similar in downregulated proteins in the OC (391>171), but not in the FC (106<156) (Fig. 1A). The comparison between FAD cases and controls was the only one that identified a variety of biological processes (BP) during Protein set enrichment analysis. Notably, FAD FC showed upregulation of BP associated to vessel proliferation (Fig. 1B), while the OC showed downregulation of BP associated to immune system and mRNA regulation (Fig. 1C).ConclusionWe have identified the OC as a critical region of FAD pathology. Proteomic analysis of isolated microvessels from this region point towards a more deleterious profile while the FC microvessel proteome indicates an earlier stage of SVD pathology.

Developing a mass spectrometric assay to measure granulin peptides in CSF for progranulin‐associated frontotemporal dementia

AbstractBackgroundPathogenic mutations in the progranulin gene (GRN) are a key cause of frontotemporal dementia (FTD), inducing a reduced biofluid concentration of the progranulin protein (PGRN). PGRN is a cysteine‐rich glycoprotein with essential roles in inflammation and lysosomal function, made up of 7 granulin peptides and 1 paragranulin. The role of these peptides is unclear, but existing data suggests they may have contradictory roles to full‐length PGRN. With the development of numerous clinical trials aiming to treat progranulin‐associated FTD (FTD‐GRN) by increasing full‐length PGRN, it is important to establish effective outcome measures to assess treatment success and further our understanding of PGRN’s biology. Here, we aimed to develop an assay to quantify granulin peptides in cerebrospinal fluid (CSF) and determine whether they contribute to the pathology of FTD‐GRN.MethodBased on previously published explorative data of endogenous CSF peptides, 12 peptides spanning the progranulin sequence, were selected for the development of targeted assays using a quadrupole Orbitrap hybrid mass spectrometer (Fusion Tribrid, Thermo). An analytical protocol was optimised involving reduction, alkylation, molecular weight cut‐off filtration and solid phase extraction, and using isotope labelled heavy standards for quantification. Additionally, tandem mass tag (TMT) proteomics was used to analyse tryptic peptides spanning granulin and paragranulin sequence regions in 248 CSF samples from the Genetic FTD initiative (GENFI) including 56 GRN mutation carriers and 76 mutation‐negative controls.ResultPreliminary results reveal the presence of three endogenous peptides in CSF, which based on sequence matching, likely represent granulin 6 and 7 alongside the paragranulin peptide. TMT results showed significantly reduced relative peptide intensity across the granulin regions in GRN carriers CSF compared to controls (p<0.0001), but no significant difference in the paragranulin region (p>0.33).ConclusionThese findings indicate that two granulins and paragranulin are quantifiable in CSF and may have key roles in progranulin biology and potentially FTD pathology. This is supported by TMT results of differential CSF paragranulin levels compared to granulins. Continuing work will quantify CSF granulin concentrations in the GENFI cohort to assess whether these peptides have key roles in FTD‐GRN’s underlying biology and as potential outcome measures in trials.

An integrated physiology and proteomics analysis reveals the response of wheat grain to low temperature stress during booting

The low temperature (LT) in spring has become one of the principal abiotic stresses that restrict the growth and development of wheat. Diverse analyses were performed to investigate the mechanism underlying the response of wheat grain development to LT stress during booting. These included morphological observation, measurements of starch synthase activity, and determination of amylose and amylopectin content of wheat grain after exposure to treatment with LT during booting. Additionally, proteomic analysis was performed using tandem mass tags (TMT). The results showed that the plumpness of wheat grains decreased after LT stress. Moreover, the activities of sucrose synthase (SuS, EC 2.4.1.13) and ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) exhibited a significant reduction, leading to a significant reduction in the contents of amylose and amylopectin. A total of 509 differentially expressed proteins (DEPs) were identified by proteomics analysis. The GO enrichment analysis showed that the protein difference multiple in the nutritional repository activity was the largest among the molecular functions, and the up-regulated seed storage protein (SSP) played an active role in the response of grains to LT stress and subsequent damage. The KEGG enrichment analysis showed that LT stress reduced the expression of DEPs such as sucrose phosphate synthase (SPS), glucose-1-phosphate adenosine transferase (glgC), and β-fructofuranosidase (FFase) in sucrose and starch metabolic pathways, thus affecting the synthesis of grain starch. In addition, many heat shock proteins (HSPs) were found in the protein processing in the endoplasmic reticulum pathways, which can resist some damage caused by LT stress. These findings provide a new theoretical foundation for elucidating the underlying mechanism governing wheat yield development after exposure to LT stress in spring.

Deep quantitative proteomics analysis of the brain vasculature in Alzheimer’s disease and related tauopathies.

AbstractBackgroundDysfunction of neurovascular unit represents a major pathological hallmark of Alzheimer’s disease (AD) and age‐related cognitive impairment. Diminished blood flow and blood‐brain barrier breakdown have been linked to the accumulation of amyloid‐b (Ab) peptide in brain parenchyma and cerebral blood vessels. Additionally, cerebrovascular dysfunction has been associated with tau pathology in primary tauopathies, including progressive supranuclear palsy (PSP) where amyloid deposition is not present. Despite the significance of brain vasculature in maintaining the proper neuronal function, the proteomic profiles of cerebrovasculature remain poorly characterized.MethodsHere, for the first time we implemented a tandem mass tag spectrometry (TMT‐MS) approach to thoroughly examine proteomic features of human cerebrovasculature from postmortem brain tissues from AD, PSP, and non‐demented control individuals (N = 20‐24 cases/diagnosis) from the University of Pennsylvania School of Medicine Brain Bank. The human cerebrovasculature was isolated from frontal cortex using density‐mediated separation to purify blood vessels from parenchymal components.ResultsThe quantification of over 9,800 proteins allowed us to generate deep TMT protein co‐expression network consisting of 93 communities of proteins displaying wide biological heterogeneity associated with diverse brain pathologies. Importantly, the comparison of vascular proteome with proteomic data obtained from bulk brain tissues from the same individuals gave us a unique opportunity to interrogate the relationship and preservation between different data sets. We also explored how candidate gene products that confer the risk for AD or PSP map to the discovered protein modules.ConclusionsIn summary, by taking advantage of our network‐driven approach we nominated pathways strongly correlated with AD‐like neuropathological traits and vascular biology that could provide the foundation for our understanding of the mechanisms governing AD‐linked breakdown of BBB, dysregulation of the cerebral blood flow, and pathological accumulation of protein aggregates in human vasculature.

Reduced Proliferation and Cell Death of Acute Myeloid Leukemia Cell Lines after Acalabrutinib Treatment

Background Acute myeloid leukemia (AML) still remains a significant challenge, with intensive chemotherapy treatments showing limited efficacy and lack of druggable targets in contrast to other hematologic malignancies. Exosomes, nanomolecules derived from cells reflect the parent cell's state, present opportunities for disease biomarker discovery and targeted therapy. Proteomic analysis of AML-derived exosomes may identify druggable targets despite limited prior research. In this study we investigated the potential of exosomes as a tool for target discovery in AML. Methods We conducted a proteomic analysis of AML-derived exosomes from HL-60, KG-1, and THP-1 cell lines, using fibroblast and mesenchymal stem cells as controls. Exosomes of each cell line were isolated, and digested via filter-aided sample preparation (FASP), followed by isobaric tagging using tandem mass tag (TMT) reagents. The samples were then fractionated using high-pH reversed-phase liquid chromatography and analyzed using Q-Exactive Orbitrap Hybrid Mass Spectrometry. Proteomic data were evaluated using SEQUEST in Proteome Discoverer 2.1 with a false discovery rate set to 1%. Differentially expressed proteins (DEPs) were identified and visualized in an interaction network and was analyzed for the gene ontology biological process (GOBP) using DAVID software. Protein-protein interactions were analyzed using the STRING software. Based on proteomic analysis, we identified the BTK-related pathway as potential druggable target for AML cells. AML cells were incubated with ACT, a BTK inhibitor, in various exposure time and density. Cell viability and proliferation rate was assessed using CCK-8 assay. BTK activity and signaling were assessed via Western blot analysis and immunocytochemistry of AML-derived exosomes. Cell death was evaluated using Annexin-V & PI apoptosis kit and FACS analysis. Statistical analysis was performed to ascertain significance. Results Proteomics of exosomes from three AML cell types revealed distinct protein profiles via LC-MS, with 2450 unique proteins identified (Figure 1A). For KG1, HL60, and THP1, the number of up-regulated DEPs were 438, 434, and 408, and the number of down-regulated DEPs was 126, 118, and 89 (fold change>1.5 or <0.67, P<0.05). Gene ontology analysis identified four main categories of biological processes, including cell migration, T-cell pathways, cell damage, and vesicle formation pathways. Investigation into protein-protein interactions using the STRING software allowed selection of tyrosine kinases SYK and LYN as biomarker candidates. Both kinases regulate BTK, a critical molecule in B-cell antigen receptor signaling. SYK and LYN were found to be enriched in exosomes, promoting phosphorylation of BTK in recipient cells. (Figure 1B) Cell viability experiments with AML cell lines revealed reduced viability upon treatment with ACT. Viability decreased with increased exposure time and density, with the highest effects observed at 1,000nM ACT concentration for 48 hours (Figure 2A). This was accompanied by decreased cell numbers and proliferation rate which was significant in the HL-60 and KG-1 cell lines (Figure 2B). Western blot analysis demonstrated significant decrease of the BTK downstream signaling (P-BTK, PKC and p-ERK) by acalabrutinib treatment (1,000nM) at 48 hours (Figure 2C). Proliferative markers, such as PCNA, and Cyclin D1 were also significantly decreased. LYN and SYK was also decreased in the exosomes of HL-60 and THP-1 cell line. The inhibition of P-BTK and Cyclin D1 by ACT was further confirmed through Immunofluorescence assay. Finally, ACT was shown to promote cell death in AML cell lines, as evidenced by an apoptosis assay, with the most significant increases observed in cells treated exposed to 1,000nM ACT for 72 h (Figure 2D). Conclusion Overall, the findings of this study revealed that acalabrutinib reduced proliferation and induced cell death in AML cells suggesting the potential of BTK inhibitors in AML treatment. This was explained through proteomic analysis of AML-derived exosomes. However, further studies are required to validate the clinical use of exosomes as biomarkers and druggable target.

Identification of a Novel AML Patient Subset Sensitive to Venetoclax Using Matched Proteomics and Diverse Ex Vivo Models

AML is one of the most common and deadly hematopoietic malignancies. The molecular and biological heterogeneity of AML poses significant challenges to the identification of novel agents to treat it and the optimal triage of patients for the most effective therapeutic regimens. Ex vivo models for drug sensitivity using cell lines and/or primary AML samples have been a fruitful resource for the exploration and discovery of novel compounds in the treatment of AML, and investigators have coupled genomic and transcriptomic profiling with ex vivo models in the hope to further enhance the reliability and accuracy of results. However, primary AML blasts are molecularly complex and fastidious, relying on extracellular matrix (ECM) and signals for optimal survival, making their use for large, high-throughput drug screens impractical. Available AML cell lines are more robust and less dependent upon the ECM but often harbor strong driver alterations (e.g., KMT2A rearrangements, TP53 mutations) that occur in only a small subpopulation of AML cases. Thus, current AML cell lines may not accurately predict agent sensitivity for most patients. Here, we report the results of a novel approach coupling cutting-edge proteomics with ex vivo drug sensitivity models to develop and describe a more diverse and representative set of patient-derived cell models for drug discovery and therapeutic positioning. Initial studies focused on proteomic biomarker signatures and cell lines to predict drug sensitivity for Venetoclax, which has become a stable component for treatment of older and unfit patients with AML. To identify a proteomic biosignature associated with Venetoclax sensitivity, we examined the Beat AML dataset of 210 patient proteomics coupled to ex vivo sensitivity assays. Beat AML patients clustered by unbiased proteomics which resulted in a cluster with a patient population significantly enriched in sensitivity to Venetoclax. A partial least squares discriminant analysis (PLSDA) was used to discover proteins driving the differentiation of Venetoclax sensitive and insensitive clusters. The resulting significant proteins describe a mechanism of altered DNA double stranded break repair and Interleukin-8 (IL-8)-CXCR2 signaling separating patients. These mechanisms were partially comprised of high-quality targets with differential analysis results shown in Fig. 1A. The identities of the targets will be revealed at the time of presentation as they are currently moving forward in preclinical studies. To examine the potential predictive power of the Venetoclax sensitivity biosignature a set of novel ex vivo cell models were derived from a diverse independent population of patients obtained from FHCC/UW Myeloid Disease Repository. This population was not enriched for KMT2A or TP53 mutations. The proteomes of 90 diagnostic AML samples were examined and measured via a tandem mass tag protocol coupled with liquid chromatography with tandem mass spectrometry (TMT-LC-MS/MS). In addition, a proprietary set of novel ex vivo AML cell lines (n=17) representing the diverse genomic phenotype were generated from this same cohort of patients using proprietary media and pyrimidoindole derivative agonists of hematopoietic stem cell self-renewal. As with the primary samples, TMT-LC-MS/MS was utilized to quantify the proteome within the proprietary AML cell lines and 16 commercially available lines. Viability assays with Venetoclax were performed for all ex vivo cell models. Ex vivo cell models were then grouped based on the Venetoclax biosignature. The correlation between the proteomic biosignature for Venetoclax the measured sensitivity to Venetoclax was then examined for the ex vivo cell models (Fig. 1B). The results showed a strong correlation between the biosignature and actual ex vivo sensitivity to Venetoclax in proprietary cell models, but not in the 16 currently available cell models. Genetic drift of cell models from the primary AML patients was examined using differential proteomic analysis and no significant drift was found. The results indicated that the primary AML specimens harbored the Venetoclax sensitivity signature. Our findings reveal that the integration of unbiased patient proteomics with optimized ex vivo cell cultures can be used to identify sensitive patient subsets and exclude non-responders for novel therapeutics, as well as expanding patient access for currently approved therapies.

Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Proteomic Analysis Based on TMT Regarding the Therapeutic Action of Rhizoma Drynariae on Rats in an Osteoporosis Model.

Primary osteoporosis has increasingly become one of the risk factors affecting human health, and the clinical effect and action mechanism of traditional Chinese medicine in the treatment of primary osteoporosis have been widely studied. Previous studies have confirmed that in traditional Chinese medicine (TCM), Drynaria rhizome has a role in improving bone density. In this study, a tandem mass tag (TMT)-based proteomic analysis was conducted to derive potential targets for Drynaria rhizome treatment in postmenopausal osteoporosis. The model group (OVX) and experimental group (OVXDF) for menopausal osteoporosis were established using the universally acknowledged ovariectomy method, and the OVXDF group was given 0.48g/kg Rhizoma Drynariae solution by gavage for 12 weeks. After 12 weeks, femurs of rats selected for this study were examined with a bone mineral density (BMD) test, Micro-CT, ELISABiochemical testing, hematoxylin and eosin (HE) staining, and immunohistochemistry. A certain portion of the bone tissue was studied with a TMT-based proteomic analysis and functional and pathway enrichment analysis. Finally, key target genes were selected for Western blotting for validation. The comparison of the OVXDF and OVX groups indicated that Drynaria rhizome could improve bone density. In the TMT-based proteomic analysis, the comparison of these two groups revealed a total of 126 differentially expressed proteins (DEPs), of which 62 were upregulated and 64 were downregulated. Further, by comparing the differential genes between the OVXDF and OVX groups and between the OVX and SHAM groups, we concluded that the 27 differential genes were significantly changed in the rats selected for the osteoporosis model after Drynaria rhizome intragastric administration. The gene ontology (GO) enrichment analysis of DEPs showed that molecular function was mainly involved in biological processes, such as glucose metabolism, carbohydrate metabolism, immune responses, and aging. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEPs revealed that multiple differential genes were enriched in the estrogen and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Relationships with nitrogen metabolism, glycerophospholipid metabolism, secretion systems, and tumor diseases were also observed. Western blotting was consistent with the analysis. We used TMT-based proteomics to analyze the positive effects of TCM Drynaria rhizome, which can regulate related proteins through the unique roles of multiple mechanisms, targets, and pathways. This treatment approach can regulate oxidative stress, improve lipid metabolism, reduce the inflammatory response mechanism, and improve bone density. These benefits highlight the unique advantages of TCM in the treatment of primary osteoporosis.

Comparison of biliary protein spectrum in gallstone patients with obesity and those with normal body weight

BackgroundObesity is a common public health issue and is currently deemed a disease. Research has shown that the risk of gallstones in individuals with obesity is elevated. This study aimed to explore the bile proteomics differences between cholelithiasis patients with obesity and normal body weight. MethodsBile samples from 20 patients (10 with obesity and 10 with normal body weight) who underwent laparoscopic cholecystectomy at our center were subjected to tandem mass tag labeling (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by further bioinformatic analysis. ResultsAmong the differentially expressed proteins, 23 were upregulated and 67 were downregulated. Bioinformatic analysis indicated that these differentially expressed proteins were mainly involved in cell development, inflammatory responses, glycerolipid metabolic processes, and protein activation cascades. In addition, the activity of the peroxisome proliferator-activated receptor (PPAR, a subfamily of nuclear receptors) signaling pathway was decreased in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Two downregulated proteins in the PPAR signaling pathway, APO A-I and APO A-II, were confirmed using enzyme-linked immunosorbent assay. ConclusionsThe PPAR signaling pathway may play a crucial role in the development of cholelithiasis among patients with obesity. Furthermore, biliary proteomics profiling of gallstones patients with obesity is revealed, providing a reference for future research.

Proteomic analysis of boar sperm with differential ability of liquid preservation at 17 °C

Understanding the liquid preservation ability of boar sperm is pivotal for efficient management and breeding of livestock. Although sperm proteins play an important role in semen quality and freezability, how the levels of protein change in boar sperm with different liquid preservation abilities at 17 °C remains unclear. In this study, two groups of boar sperm with extreme difference in liquid preservation ability, namely the good preservation ability (GPA) and the poor preservation ability (PPA) groups, were selected by evaluating sperm motility parameters on the 7th day of liquid preservation at 17 °C. Quantitative proteomics based on tandem mass tag (TMT) labeling was used, sperm proteomic characteristics from two groups were analyzed, and potentially key proteins related to the fluid preservation ability of sperm were identified. A total of 187 differentially expressed proteins (DEPs) were identified among 2791 quantified proteins, including 85 upregulated, and 102 downregulated proteins. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEPs revealed that they were enriched in GO terms associated with response to oxidative stress, enzyme activity related to oxidative stress or redox reactions, and several metabolic activities. The significant KEGG pathways included peroxisome, metabolic pathways, selenocompound metabolism, and collection duct acid secretion. In addition, analysis of protein–protein interactions further identified 8 proteins that could be used as biomarker candidates, including GPX5, GLRX, ENO4, QPCT, BBS7, OXSR1, DHRS4 and AP2S1, which may play an essential role in indicating the liquid preservation ability of boar sperm. These findings in this study provide new insights into the underlying molecular mechanisms of the liquid preservation ability of boar sperm. Moreover, the selected candidate proteins can serve as a reference for evaluating sperm quality or preservation ability in boars and their application in related biotechnologies.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

Simple Tool for Rapidly Assessing the Quality of Multiplexed Single Cell Proteomics Data.

Recent advances in the sensitivity and speed of mass spectrometers coupled with improved sample preparation methods have enabled the field of single cell proteomics to proliferate. While heavy development is occurring in the label free space, dramatic improvements in throughput are provided by multiplexing with tandem mass tags. Hundreds or thousands of single cells can be analyzed with this method, yielding large data sets which may contain poor data arising from loss of material during cell sorting or poor digestion, labeling, and lysis. To date, no tools have been described that can assess data quality prior to data processing. We present herein a lightweight python script and accompanying graphic user interface that can rapidly quantify reporter ion peaks within each MS/MS spectrum in a file. With simple summary reports, we can identify single cell samples that fail to pass a set quality threshold, thus reducing analysis time waste. In addition, this tool, Diagnostic Ion Data Analysis Reduction (DIDAR), will create reduced MGF files containing only spectra possessing a user-specified number of single cell reporter ions. By reducing the number of spectra that have excessive zero values, we can speed up sample processing with little loss in data completeness as these spectra are removed in later stages in data processing workflows. DIDAR and the DIDAR GUI are compatible with all modern operating systems and are available at: https://github.com/orsburn/DIDARSCPQC. All files described in this study are available at www.massive.ucsd.edu as accession MSV000088887.

Identification of a Candidate Gene Regulating Intramuscular Fat Content in Pigs through the Integrative Analysis of Transcriptomics and Proteomics Data.

Pork is a widely consumed source of animal protein worldwide, and the intramuscular fat (IMF) content in pork plays a crucial role in determining its quality. In this study, we sought to identify candidate genes that regulate IMF deposition in pigs. We performed tandem mass tags (TMT)-based quantitative proteomics analysis using Longissimus dorsi (LD) muscle samples obtained from eight pigs with extremely high and low IMF content among a group of 28 Duroc pigs and identified 50 differentially abundant proteins (DAPs). Additionally, we compared the proteomics data with RNA-sequencing data obtained in our previous study and identified TUSC5 as a differentially expressed gene corresponding to the relevant DAP. To investigate the potential role of TUSC5 in adipogenesis, we suppressed TUSC5 expression in mouse 3T3-L1 preadipocytes using short hairpin RNA (shRNA) and observed a significant reduction in the differentiation of 3T3-L1 cells into adipocytes, as indicated by Oil Red O staining and triglyceride content. Moreover, we observed a reduction in the expression of genes associated with adipogenesis (PPARG, CEBPA, FABP4, and FASN) following TUSC5 suppression. Through an integrative analysis of transcriptomics and proteomics data, our study identified TUSC5 as a crucial candidate gene associated with the regulation of IMF content in pigs.

ZnO nanoparticles impair autophagic flux and cell viability through the TRIM16-NRF2-p62 pathway in inflammatory keratinocytes

PurposeZinc oxide nanoparticles (ZnO NPs) are widely used in sunscreen, cosmetics, and topical drugs. Most previous studies have confirmed the safety of ZnO NPs applied to normal skin; however, little is known about the safety and potential toxicity of ZnO NPs applied to inflamed skin. This study aimed to evaluate the exposure risk of ZnO NPs in the treatment of inflammatory skin diseases. MethodsNormal human and tumor necrosis factor-α (TNF-α)-induced inflammatory keratinocytes were incubated with ZnO NPs to assess their toxic effects on cell viability and autophagy signaling pathway. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of inflammatory keratinocytes with ZnO NPs. Protein expression was assessed by Western blot, and double fluorescent labeling and siRNA-knockdown further elucidated the role of the TRIM16-NRF2-p62 pathway in mediating the effects of ZnO NP. ResultsIn TNF-α-induced inflammatory keratinocytes, ZnO NPs activated cytoprotective autophagy and mediated p62-related autophagic flux block, thereby reducing the viability of inflammatory keratinocytes. Additionally, TRIM16-NRF2 was essential in ZnO NP-mediated autophagy flux block and cell viability reduction in inflammatory keratinocytes. Inhibition of the TRIM16-NRF2 pathway reduced p62 levels, alleviated autophagy flux blockade, and slightly restored the viability of inflammatory keratinocytes. ConclusionZnO NPs activated protective cell autophagy. Blockade of autophagy flux mediated by the TRIM16-NRF2-p62 pathway led to decreased cell viability. This study provided a deeper understanding of the toxicity mechanism of ZnO NPs in inflammatory keratinocytes.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H2S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Acute severe hypoglycemia alters mouse brain microvascular proteome

Hypoglycemia increases the risk related to stroke and neurodegenerative diseases, however, the underlying mechanisms are unclear. For the first time, we studied the effect of a single episode (acute) of severe (ASH) and mild (AMH) hypoglycemia on mouse brain microvascular proteome. After four-hour fasting, insulin was administered (i.p) to lower mean blood glucose in mice and induce ∼30 minutes of ASH (∼30 mg/dL) or AMH (∼75 mg/dL), whereas a similar volume of saline was given to control mice (∼130 mg/dL). Blood glucose was allowed to recover over 60 minutes either spontaneously or by 20% dextrose administration (i.p). Twenty-four hours later, the brain microvessels (BMVs) were isolated, and tandem mass tag (TMT)–based quantitative proteomics was performed using liquid chromatography-mass spectrometry (LC/MS). When compared to control, ASH significantly downregulated 13 proteins (p ≤ 0.05) whereas 23 proteins showed a strong trend toward decrease (p ≤ 0.10). When compared to AMH, ASH significantly induced the expression of 35 proteins with 13 proteins showing an increasing trend. AMH downregulated only 3 proteins. ASH-induced downregulated proteins are involved in actin cytoskeleton maintenance needed for cell shape and migration which are critical for blood-brain barrier maintenance and angiogenesis. In contrast, ASH-induced upregulated proteins are RNA-binding proteins involved in RNA splicing, transport, and stability. Thus, ASH alters BMV proteomics to impair cytoskeletal integrity and RNA processing which are critical for cerebrovascular function.

Temporal proteomic and phosphoproteomic profiling of the left ventricle in a novel rat model of ischemia/reperfusion with or without ischemic preconditioning

Abstract Background The relationship between ischemic preconditioning (IPC) and myocardial stunning is incompletely understood. Preconditioning has been shown to protect against ischemic injury, whereas myocardial stunning, i.e. transient contractile dysfunction in the setting of ischemia, can lead to severe heart failure and has been considered a deleterious process. To understand this relationship, we recently developed a novel rat model of ischemia-reperfusion (I/R) injury in the presence or absence of IPC. In the presence of IPC (2 cycles of 5 min of I/R followed by 13.5 min of ischemia), we observe a profound myocardial stunning at 4h of reperfusion which completely resolves at 48h. In the absence of IPC, we observe necrosis at 4h of reperfusion and sustained injury at 48h. Despite extensive studies on IPC and myocardial stunning in the setting of I/R, the molecular basis of the relationship between these phenomena are still unknown. Purpose To investigate alterations in protein expression after IPC and myocardial stunning in a novel rat model of I/R injury. Methods We utilized tandem mass tags and mass spectrometry to identify temporal changes in the proteomic- and phosphoproteomic-profiles in left ventricular (LV) tissue from rat hearts following 13.5 min of ischemia and 4h of reperfusion in the presence of absence of IPC. Results Proteomics analysis of the LV tissues collected at baseline showed that 115 proteins were differentially expressed between IPC and non-IPC groups. We observed abundant changes in sarcoplasmic proteins, sarcoplasmic reticulum-related proteins, and actin-based cell projection proteins. Most of these proteins are implicated in arylesterase activity, haemoglobin binding, and nitrogen metabolism. At 4h, differentially expressed proteins were predominantly related to wound healing, coagulation, and lipase activity. Phosphoproteomic analysis at baseline showed that 686 phosphosites were differentially regulated between IPC and non-IPC groups. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins, Z-disc, I-band, contractile fiber associated with actin binding, and the thyroid hormone signalling pathway. At 4h, 520 phosphosites differed between IPC and non-IPC rats; these phosphosites were abundant in contractile fiber proteins, myofibril proteins, and sarcomere proteins associated with calcium signalling and cardiomyopathies. Conclusions This study demonstrates marked changes in proteins and phosphosites related to lipase activity, haemoglobin binding, calcium signalling, and the cardiac contractile apparatus in IPC versus non-IPC groups. This extensive catalogue of novel proteins and phosphosites associated with IPC and myocardial stunning in the setting of I/R injury sets the stage for future manipulation of myocardial stunning and preconditioning in both experimental and clinical settings.

Abstract 13568: Proteomic Analysis Reveals Protein Alterations in Drp1 Cardiac Knockout Mice With Circadian Rhythm Disruption

Introduction: Mitochondria dynamics are altered in circadian rhythm disruption, and dynamin-related protein 1 (Drp1) may be an important target. Methods: Wild-type and Drp1 cardiac-specific knockout mice were divided into normal circadian group and circadian disorder group, respectively. Normal circadian group mice were housed in a standard environment (12 hours of light/12 hours of darkness), while dark group mice were housed in a 24-hour dark environment. After 4 weeks, the cardiac tissues were analysed using Tandem Mass Tag proteomics. Differentially expressed proteins (DEPs) was identified. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Gene set enrichment analysis (GSEA) was performed to find enriched biological processes. Results: Compared with the WT/N group, the Drp1-/N group possessed a total of 176 DEPs. These proteins are widely involved in mitochondrial membrane and metabolism process. We identified several differentially expressed mitochondrial dynamics-related genes, including Drp1, using Western blotting. Compared with the WT/D group, the Drp1-/D group exhibited a total of 126 DEPs. GO and KEGG showed that the DEPs were mainly involved in response to stress, metabolic pathways. For Drp1 cKO mice, the protein changes caused by the circadian rhythm disorder were mainly concentrated in vitamin transport, complement coagulation cascades. Conclusions: This study may provide an insight into the myocardial protein changes caused by Drp1 cKO with circadian rhythm disruption, which may involve in terms such as metabolic pathways, complement coagulation, and vitamin transport.