Proteome Discoverer Research Articles

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Serum proteomics for the identification of biomarkers to flag predilection of COVID19 patients to various organ morbidities

BackgroundCOVID19 is a pandemic that has affected millions around the world since March 2020. While many patients recovered completely with mild illness, many patients succumbed to various organ morbidities. This heterogeneity in the clinical presentation of COVID19 infection has posed a challenge to clinicians around the world. It is therefore crucial to identify specific organ-related morbidity for effective treatment and better patient outcomes. We have carried out serum-based proteomic experiments to identify protein biomarkers that can flag organ dysfunctions in COVID19 patients.MethodsCOVID19 patients were screened and tested at various hospitals across New Delhi, India. 114 serum samples from these patients, with and without organ morbidities were collected and annotated based on clinical presentation and treatment history. Of these, 29 samples comprising of heart, lung, kidney, gastrointestinal, liver, and neurological morbidities were considered for the discovery phase of the experiment. Proteins were isolated, quantified, trypsin digested, and the peptides were subjected to liquid chromatography assisted tandem mass spectrometry analysis. Data analysis was carried out using Proteome Discoverer software. Fold change analysis was carried out on MetaboAnalyst. KEGG, Reactome, and Wiki Pathway analysis of differentially expressed proteins were carried out using the STRING database. Potential biomarker candidates for various organ morbidities were validated using ELISA.Results254 unique proteins were identified from all the samples with a subset of 12–31 differentially expressed proteins in each of the clinical phenotypes. These proteins establish complement and coagulation cascade pathways in the pathogenesis of the organ morbidities. Validation experiments along with their diagnostic parameters confirm Secreted Protein Acidic and Rich in Cysteine, Cystatin C, and Catalase as potential biomarker candidates that can flag cardiovascular disease, renal disease, and respiratory disease, respectively.ConclusionsLabel free serum proteomics shows differential protein expression in COVID19 patients with morbidity as compared to those without morbidity. Identified biomarker candidates hold promise to flag organ morbidities in COVID19 for efficient patient care.

B-146 Unlocking the potential of large-cohort proteomics studies with the Orbitrap Astral mass platform

Abstract Background Large-cohort proteomics analysis using mass spectrometry is a powerful approach to discover and validate new biomarkers. In combination with clinical data and computational analysis, large-cohort proteomics study brings opportunities in improving early diagnosis, refining patient stratification, and predicting/monitoring treatment response. Yet, to achieve meaningful biological insights in large-cohort studies, robust, reproducible, and comprehensive proteome profiling in a high-throughput manner still remains challenging. Here, we use a high-resolution accurate mass (HRAM) Oribtrap Astral platform to enable high-quality and robust protein quantification across thousands of LC-MS/MS analyses. With a throughput of 100 samples/day, we can reproducibly profile ∼9000 proteins from human cell line and ∼800 proteins from undepleted plasma across multiple instruments and more than 10 consecutive days in a 24/7 operation mode. Methods Experiments were performed on multiple Orbitrap Astral mass spectrometers with and without FAIMS Pro interface. Chromatographic separations were performed using a trap/elute method on Vanquish Neo system at a 11-minute gradient and a flow rate of 1ul/min, resulting in a throughput of 100 samples/day. Undepleted plasma digest was analyzed in a 24/7 mode for > 1000 injections on each LC-MS/MS setup. In addition, HeLa digest as quality control (QC) was analyzed every 12hours. The Oribtrap Astral platform was operated in data-independent acquisition (DIA) mode using a full scan with m/z 380-980, and DIA MS/MS scans with an isolation window of 2Th. Resulting DIA raw files were automatically transferred and processed using Chimerys in a beta version of Proteome Discoverer software. Results Multiple LC-MS/MS systems were operated in DIA mode either with or without FAIMS Pro device in a 24/7 operation mode at a throughput of 100SPD. Undepleted plasma digest was analyzed with >1000 injections on each LC-MS/MS setup. To monitor the baseline performance, HeLa digest served as QC were analyzed periodically every 12 hours with 3 technical replicates each time. To effectively manage and analyze these thousands of data files generated, we developed an automated data transfer and analysis pipeline. Automatically, the resulting DIA raw data files were immediately transferred to a server, then processed by state-of-the-art intelligent search algorithm Chimerys. Benefiting from the ultra-high scan speed of up to 200Hz on the Orbitrap Astral mass spectrometer, a much narrower isolation window width of 2Th was applied in the DIA method, comparing to 10-20Th on the classic DIA method. As a result, ∼9000 proteins from HeLa digest and ∼800 proteins from undepleted plasma digest were identified within only a 11-minutes gradient, respectively. More than 80% of the plasma proteins were reproducibly identified and quantified from all the runs on each LC-MS/MS setup, indicating a great reproducibility from run-to-run longitudinally. Stable and robust peptide quantitation was observed by extracting peptides with high, medium, and low abundant across the runs. Importantly, QC showed no performance degradation throughout the entire study, indicating high robustness of the entire LC-MS/MS setup. Conclusions Orbitrap Astral mass platform can comprehensively analyze the proteome of >1000s of sample robustly and reproducibly in a high-throughput manner, addressing the needs in large-cohort studies.

A pilot study on the differential urine proteomic profile of subjects with community-acquired acute kidney injury who recover versus those who do not recover completely at 4 months after hospital discharge.

Community-acquired acute kidney injury (CA-AKI) is a sudden structural damage and loss of kidney function in otherwise healthy individuals outside of hospital settings having high morbidity and mortality rates worldwide. Long-term sequelae of AKI involve an associated risk of progression to chronic kidney disease (CKD). Serum creatinine (SCr), the currently used clinical parameter for diagnosing AKI, varies greatly with age, gender, diet, and muscle mass. In the present study, we investigated the difference in urinary proteomic profile of subjects that recovered (R) and incompletely recovered (IR) from CA-AKI, 4 months after hospital discharge. Study subjects were recruited from ongoing study of CA-AKI cohort. Patients with either sex or age > 18 years with no underline CKD were enrolled at the time of hospital discharge. Incomplete recovery from CA-AKI was defined as eGFR < 60 mL/min/1.73 m2 or dialysis dependence at 4 months after discharge. Second-morning urine samples were collected, and proteome analysis was performed with LC-MS/MS. Data were analyzed by Proteome Discoverer platform 2.2 (Thermo Scientific) using statistical and various bioinformatics tools for abundance of protein, cellular component, protein class and biological process were analyzed in the recovered and incompletely recovered groups. A total of 28 subjects (14 in each group) were enrolled. Collectively, 2019 peptides and proteins with 30 high-abundance proteins in the incompletely recovered group (R/IR <0.5, abundance ratio adj. p-value <0.05) and 11 high-abundance proteins in the incompletely recovered group (R/IR >2.0, abundance ratio adj. p-value <0.05) were identified. Tissue specificity analysis, GO enrichment analysis, and pathway enrichment analysis revealed significant proteins in both the groups that are part of different pathways and might be playing crucial role in renal recovery during the 4-month span after hospital discharge. In conclusion, this study helped in identifying potential proteins and associated pathways that are either upregulated or downregulated at the time of hospital discharge in incompletely recovered CA-AKI patients that can be further investigated to check for their exact role in the disease progression or repair.

Investigating the Neuroprotective Effect of Withaferin-A in a Parkinsonian Mouse Model

Parkinson's disease (PD) is an enduring and advancing neurodegenerative disorder that impacts millions worldwide. While options to manage its symptoms are available, a definitive cure remains elusive. To enhance the quality of life for PD patients and to alleviate symptoms, various classes of medications are employed including Levodopa (L-Dopa), Dopamine Agonists and Dopamine Agonists. Within this investigation, we investigated the neuroprotective potential of Withaferin-A within a mouse model of Parkinsonism, utilizing a proteomics methodology. Withaferin A, a natural compound extracted from the plant Withania somnifera, colloquially known as ashwagandha, has been explored for its therapeutic attributes across several conditions including PD. To assess the neuroprotective capacity of Withaferin A (WA), we employed a quantitative proteomics strategy to pinpoint proteins with distinct expression patterns in mouse models of Parkinson's disease (PD) treated with WA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The experimental setup encompassed four groups of mice: Group I as control, group II administered MPTP, group III treated with Withaferin A and group IV receiving Withaferin A treatment. After humane termination and intracardial infusion, the substantia nigra (SN) region of the mice brains was collected. Employing tandem mass tags, quantitative proteomics was executed on the obtained brain samples through an Orbitrap Fusion Mass spectrometer (MS). The subsequent analysis of LC-MS/MS data was conducted via Proteome Discoverer and NCBI's mouse protein database using the MASCOT search engine. This comprehensive exploration unveiled a list of 6044 proteins among which alpha-synuclein was identified. Notably, 133 proteins exhibited differential expression. These differentially expressed proteins played pivotal roles in significant biological processes including cellular processes (signal transduction), metabolic processes (NADP metabolic process) and signaling (dopamine receptor signaling). Crucial pathways uncovered encompass Parkinson's disease and the dopamine receptor-mediated signaling pathway. It is our belief that the insights gleaned from this study provide a foundation for formulating targeted treatment strategies to combat Parkinson's disease, thus potentially enhancing the therapeutic landscape for individuals affected by this condition.

Sika Deer Velvet Antler Peptide Exerts Neuroprotective Effect in a Parkinson's Disease Model via Regulating Oxidative Damage and Gut Microbiota.

Parkinson's disease (PD) is the second most common neurodegenerative disorder globally. Recognizing the potential of velvet antler in the nervous system, as shown in numerous studies, this research was aimed at evaluating the neuroprotective effects of Sika Deer velvet antler peptide (VAP), along with the underlying mechanisms in neurotoxin-induced PD models. Initially, a peptidomic analysis of the VAP, which comprised 189 varieties of peptides, was conducted using LC-MS. Nine sequences were identified as significant using Proteome Discoverer 2.5 software. In a cellular model of PD, where PC12 cells are treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the administration of the VAP reduced the cell damage and apoptosis induced by MPP+. This protective effect was associated with a decrease in oxidative stress. This protective mechanism was found to be mediated through the activation of the SIRT1-dependent Akt/Nrf2/HO-1-signaling pathway. In animal models, specifically in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, the administration of the VAP effectively reduced the dopaminergic neuron damage and reversed the neurobehavioral deficits. They also diminished microglia activation and apoptosis, all without any noticeable adverse effects. Additionally, the VAP was observed to beneficially alter the gut microbiota, as marked by an increase in the abundances of Prevotellaceae, Helicobacteraceae, and Prevotella. These findings suggest that VAP exerts its neuroprotective effect against neurodegeneration by inhibiting oxidative stress and modulating gut microbiota.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

O-267 Extracellular vesicles as biomarkers of endometriosis

Abstract Study question Can extracellular vesicles serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound? Summary answer Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the diagnosis of peritoneal lesions currently relies on invasive laparoscopy, which leads to a delay of 8-10 years. We have previously shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis specific EVs. Traced in peripheral blood (PB), EVs could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration Women 18-45 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study (HREC 20-159A/HREC 20-882A). Following informed consent, we collected PF and PB samples from n = 101 participants (n = 47 controls, n = 54 cases). Exclusion criteria were pregnancy, malignancy, and menopause. Participants/materials, setting, methods We processed PF and PB samples by differential ultracentrifugation and validated the presence of EVs by nanoparticle tracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). EVs were analysed by untargeted, label-based quantitative proteomics using Tandem Mass Tags (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher) followed by statistical analysis using R. Candidate biomarker proteins were tested by traditional and automated Western blotting (WES, ProteinSimple). Main results and the role of chance We ascertained the identity of EVs by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomic analysis identified 9145 protein groups across all sample groups, with 602 significantly regulated between comparisons (adjusted P-value &amp;lt;0.05). In PF, 533 proteins changed significantly in abundance (245 up/288 down), while in blood, only four proteins changed in abundance. We tested the presence/absence of two of these as candidate markers on PB EV protein preparations from n = 32 samples to date; our best marker showed a sensitivity of 67% and a specificity of 90%; upon exclusion of severe endometriosis cases, sensitivity improved to 80%. Limitations, reasons for caution Because of the changes in vesicle composition and amounts with the menstrual cycle, we have to group the samples by menstrual cycle phase and disease severity. This leaves relatively small numbers per group per condition. Another limitation concerns the relatively small amount of sample liquid and thus, EVs available for analysis. Wider implications of the findings Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. A non-invasive clinical test would allow for earlier diagnosis and thus treatment of endometriosis. It would also allow patients to initiate fertility treatment early. Trial registration number not applicable

A prospective study of the proteome of equine pre-implantation embryo.

The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize invitro fertilization techniques.

Proteomic investigation of acute and chronic hypoxia/reoxygenation responsive proteins and pathways in H9C2 cardiomyoblasts.

Ischemic heart diseases continue to be a significant global cardiovascular problem in today's world. Myocardial reperfusion (R) is provided with an effective and rapid treatment; however, it can lead to fatal results, as well as ischemia (I). This study aims to use proteomic analysis to assess proteins and pathways in H9C2 cardiomyoblast cells exposed to hypoxic conditions, followed by reoxygenation, representing I/R injury for both short and long terms, reflecting acute and chronic hypoxia, respectively. Utilizing advanced techniques, our goal is to identify and characterize key proteins undergoing alterations during these critical phases. H9C2 cardiomyoblasts, a commonly used cell line for simulating in vivo I/R damage, were exposed to normoxia and hypoxia (0.4% O2) in six experimental groups: normoxia (3h), acute hypoxia (3h), acute hypoxia (3h) + reoxygenation (3h), normoxia (21h), chronic hypoxia (21h), and chronic hypoxia (21h) + reoxygenation (3h). Analyses were conducted using Nano LC/MSMS from tryptic digest of the whole cell lysates. Proteins were quantified using the label-free quantification (LFQ) algorithm in Proteome Discoverer 2.4. Proteomic analysis resulted in identification of 2383 protein groups. Proteins that differentially expressed in the various groups were identified (p < 0.05 among mean values for groups). Short-term hypoxia induces mitochondrial damage, energy demand, and cytoskeletal modifications. Chronic hypoxia triggers metabolic shifts, stress-response proteins, and extracellular matrix alterations. Data are available via ProteomeXchange with identifier PXD047994. Our research provides in-depth insights into how H9C2 cardiomyoblasts respond to both short-term and prolonged oxygen deprivation. Understanding hypoxia-related pathophysiology provides avenues for therapeutic intervention in hypoxia-related disorders.

Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

Clinical phosphoproteomics reveals aberrant signalling pathways and kinases in treatment-resistant cervical cancer.

e17529 Background: Cisplatin-based concurrent chemoradiation therapy (CCRT) is the standard for locally advanced cervical cancer. While cisplatin stands out as a highly effective chemotherapeutic option for cervical cancer, resistance to the drug often triggers relapses. Moreover, previous studies have highlighted the detrimental impact of cisplatin's toxicity, which extends beyond treatment efficacy to influence the overall prognosis of patients. Combination treatment with drugs targeting different molecular pathways increases the effectiveness of the treatment by disrupting multiple critical processes that cancer cells rely on for survival. Herein, we analyzed the phosphoproteomic profile of cervical cancer patients resistant to concurrent chemoradiotherapy to identify the crucial kinases responsible for therapeutic resistance. Methods: We used mass spectrometry for phosphoproteomic analysis to study signaling pathways and dysregulated kinases in CCRT resistance. Biopsy samples collected pre-therapy were processed by protein extraction, digestion, and labeling with 10-plex TMT. Peptides were fractionated by basic pH liquid chromatography, enriched using IMAC, and analyzed on an Orbitrap-Fusion-Tribrid mass spectrometer. Data analysis was performed using Proteome Discoverer and various bioinformatics and statistical methods. Results: The phosphoproteomic analysis revealed distinct phosphorylation signatures between the responders and non-responders. In concordance with the phosphoproteomic data the expression of phospho-SMC1A and CSNK2A1 is preserved in non-responder cohort of cervical cancer patients in concordance with the phosphoproteomic data. Further, the pathway analysis showed elevated presence of the DNA repair pathway in the treatment-resistant cohort. Furthermore, dysregulation in the phosphorylation of key DNA repair proteins, such as SMC1A, HMGN1, MGMT, DDB2, and MSH6, suggests their involvement in treatment. The kinase prediction analysis suggested the activation of kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM in non-responders. Subsequently, cell line based functional experiments indicated the potential of CSNK2A1 inhibitor silmitasertib in sensitizing the cells to cisplatin treatment. Conclusions: Phosphoproteomicanalysis of the cervical cancer revealed distinct phosphorylation signature and kinases involved in treatment resistance. Our findings indicated that dysregulated phosphorylation and increased expression of the kinase CSNK2A1 play a pivotal role in the treatment response of cervical cancer patients. Moreover, CSNK2A1 can sensitizes the cells to cisplatin uncovering the potential of combination treatment strategies. Our study uncovers the potential of clinical phosphoproteomics to design tailored treatment strategies in patients resistant to standard treatments.

Plasma Proteins Associated with COVID-19 Severity in Puerto Rico.

Viral strains, age, and host factors are associated with variable immune responses against SARS-CoV-2 and disease severity. Puerto Ricans have a genetic mixture of races: European, African, and Native American. We hypothesized that unique host proteins/pathways are associated with COVID-19 disease severity in Puerto Rico. Following IRB approval, a total of 95 unvaccinated men and women aged 21-71 years old were recruited in Puerto Rico from 2020-2021. Plasma samples were collected from COVID-19-positive subjects (n = 39) and COVID-19-negative individuals (n = 56) during acute disease. COVID-19-positive individuals were stratified based on symptomatology as follows: mild (n = 18), moderate (n = 13), and severe (n = 8). Quantitative proteomics was performed in plasma samples using tandem mass tag (TMT) labeling. Labeled peptides were subjected to LC/MS/MS and analyzed by Proteome Discoverer (version 2.5), Limma software (version 3.41.15), and Ingenuity Pathways Analysis (IPA, version 22.0.2). Cytokines were quantified using a human cytokine array. Proteomics analyses of severely affected COVID-19-positive individuals revealed 58 differentially expressed proteins. Cadherin-13, which participates in synaptogenesis, was downregulated in severe patients and validated by ELISA. Cytokine immunoassay showed that TNF-α levels decreased with disease severity. This study uncovers potential host predictors of COVID-19 severity and new avenues for treatment in Puerto Ricans.

Unveiling Actin Cytoskeleton Role in Mediating Chikungunya-Associated Arthritis: An Integrative Proteome-Metabolome Study.

Background: Chikungunya is a zoonotic disease caused by the Chikungunya virus (CHIKV), primarily transmitted to humans through infected Aedes mosquitoes. The infection is characterized by symptoms such as high fever, musculoskeletal pain, polyarthritis, and a rash, which can lead to severe complications such as encephalitis, meningitis, and even fatalities. While many disease manifestations resemble those of other viral infections, chronic arthritis caused by CHIKV is unique, and its molecular mechanisms remain ill-defined. Materials and Methods: Proteomics data from both cellular and patient levels of CHIKV infection were curated from PubMed and screened using inclusion and exclusion criteria. Patient serum proteomics data obtained from P RIDE underwent reanalysis using Proteome Discoverer 2.2. Enrichment and protein-protein interaction network analysis were conducted on differentially expressed proteins from both serum and cellular datasets. Metabolite data from CHIKV-infected patients were further retrieved, and their protein binding partners were identified using BindingDB. The protein-metabolite interaction pathway was further developed using MetaboAnalyst. Results: The proteomics data analysis revealed differential expression of proteins involved in critical host mechanisms, such as cholesterol metabolism and mRNA splicing, during CHIKV infection. Consistent upregulation of two actin cytoskeleton proteins, TAGLN2 and PFN1, was noted in both serum and cellular datasets, and their upregulations are associated with arthritis. Furthermore, alterations in purine metabolism were observed in the integrative proteome-metabolome analysis, correlating with cytoskeletal remodelling. Conclusion: Collectively, this integrative view sheds light on the involvement of actin cytoskeleton remodeling proteins and purine metabolic pathways in the development of arthritis during CHIKV infection.

Gentisic acid exerts neuroprotective effects in neurotoxin-induced Parkinson's disease model in zebrafish: Cross-talk between pathways related with neurodegeneration in the gut-brain axis

Given that global prevalence of Parkinson's disease (PD) is expected to rise over the next few decades, understanding the mechanisms and causes of PD is critical. With emphasis on gut-brain axis, we sought to assess the impact of gentisic acid (GA), a diphenolic compound generated from benzoic acid, in rotenone (Rot) induced PD model in zebrafish. For thirty days, adult zebrafish were exposed to GA and rotenone. Tox-Track program was used to analyze locomotor behaviors in the control, GA, Rot, and Rot + GA groups. LC-MS/MS was performed in brain and intestinal tissues. Proteome Discoverer 2.4 was used to analyze raw files, peptide lists were searched against Danio rerio proteins. Protein interactions or annotations were obtained from STRING database. Tyrosine hydroxylase (Th) staining was performed immunohistochemically in the brain. PD-related gene expressions were determined by RT-PCR. Lipid peroxidation, nitric oxide, superoxide dismutase, glutathione S-transferase, and acetylcholinesterase were measured spectrophotometrically. Improved locomotor behaviors were observed by GA treatment in Rot group as evidenced by increased average speed, exploration rate, and total distance. 5214 proteins were identified in intestinal tissues, 4114 proteins were identified in brain by LC-MS/MS. Rotenone exposure altered protein expressions related to oxidative phosphorylation in brain and intestines. Protein expressions involved in ferroptis and actin cytoskeleton changed in brain and intestines. Altered protein expressions were improved by GA. GA ameliorated Th-immunoreactivity in brain, improved park2, park7, pink1, and lrrk2 expressions. Our results show that GA may be a candidate agent to be evaluated for its potential protective effect for PD.

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SignificanceCiliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Aqueous humour protein dysregulation in Asian eyes with primary open angle glaucoma

The pathophysiology of Primary Open Angle Glaucoma (POAG) remains poorly understood. Through proteomic analysis of aqueous humour (AH) from POAG patients, we aim to identify changes in protein composition of these samples compared to control samples.High resolution mass spectrometry-based TMT6plex quantitative proteomics analysis is performed on AH samples collected from POAG patients, and compared against a control group of patients with cataracts. Data are available via ProteomeXchange with identifier PXD033153.1589 proteins were quantified from the aqueous samples using Proteome Discoverer version 2.2 software. Among these proteins, 210 were identified as unique master proteins. The proteins which were up or down-regulated by ±3 fold-change were considered significant. Human neuroblastoma full-length cDNA clone CS0DD006YL02 was significantly upregulated in patients with severe POAG on >2 medications, while actin, cytoplasmic 1, V2-7 protein (fragment), immunoglobulin-like polypeptide 1 and phosphatidylethanolamine-binding protein 4 were only present in these patients with severe POAG on >2 medications.Beta-crystallin B1 and B2, Gamma-crystallin C, D and S were significantly downregulated in the severe POAG ≤2 glaucoma medications group.Beta-crystallin B2, Gamma-crystallin D and GCT-A9 light chain variable region (fragment) were significantly downregulated in the non-severe POAG group.Actin, cytoplasmic 1 was significantly upregulated in subjects with severe POAG who required more than 2 glaucoma medications. Crystallins (Beta-crystallin B1 and B2, Gamma-crystallin C, D and S) were significantly downregulated in subjects with severe POAG who required less than 2 glaucoma medications.

Fast and Accurate Disulfide Bridge Detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography – High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools

BackgroundTandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. MethodsThe major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. ResultsA reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. ConclusionUsing this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

Data notes on the proteomics of Dendrobium huoshanense under pb treatment.

Pb stress has a negative impact on plant growth by interfering with photosynthesis and releasing reactive oxygen species, causing major risks such as heavy metal ion accumulation in the soil matrix. A proteomics experiment was conducted to determine whether protein levels of Dendrobium huoshanense changed in response to Pb stress seven to fifteen days after being sprayed with a 200mg/L Pb (NO3)2 solution. The proteomic data we gathered provides a model for investigations into the mechanisms underlying Dendrobium plant resistance to heavy metal stress. A label-free quantitative proteomics approach was employed to examine the variations in protein expression levels of D. huoshanense at different times of Pb(NO3)2 treatment. We submitted the raw data obtained from these proteomics sequencing experiments to the ProteomeXchange database with the accession number PXD047050. 63,194 mass spectra in total were compared after being imported into the Proteome Discoverer software for database search. A total of 12,402 spectral peptides were identified with a confidence level exceeding 99%, which resulted in the identification of 2,449 significantly differential proteins. These proteins can be utilized for screening, functional annotation, and enrichment analysis of differentially expressed proteins before and after heavy metal treatment experiments.