Nanoscale Liquid Chromatography Research Articles

Treatment of Refractory Mucosal Leishmaniasis Is Associated with Parasite Overexpression of HSP70 and ATPase and Reduced Host Hydrogen Peroxide Production (Brief Report).

Mucosal leishmaniasis (ML) is a deforming type of American Tegumentary Leishmaniasis caused by Leishmania (Viannia) braziliensis that frequently does not respond to treatment. Despite its relapsing clinical course, few parasites are usually found in mucosal lesions. Host and parasite factors may be responsible for this paradox in the pathogenesis of the disease, allowing for both a low parasite burden and the inability of the host to clear and eliminate the disease. In this work, we present a clinical case of relapsing ML that was treated for 25 years without success with SbV, N-methyl glucamine, sodium stibogluconate, amphotericin B deoxycholate, gabromycin, antimonial plus thalidomide, liposomal amphotericin B, Leishvacin (a vaccine made in Brazil) and miltefosine. In a comparative analysis using nanoscale liquid chromatography coupled with tandem mass spectrometry of protein extracts of L. (V.) braziliensis promastigotes isolated from the patient and from the reference strain (MHOM/BR/94/M15176), we observed increases in ATPase and HSP70 protein levels in the parasite. We also observed an impairment in the production of hydrogen peroxide by peripheral mononuclear blood monocytes (PBMCs), as assessed by the horseradish peroxidase-dependent oxidation of phenol red. We hypothesise that these parasite molecules may be linked to the impairment of host parasiticidal responses, resulting in Leishmania persistence in ML patients.

Hyphenation of microflow chromatography with electrospray ionization mass spectrometry for bioanalytical applications focusing on low molecular weight compounds: A tutorial review.

Benefits of miniaturized chromatography with various detection modes, such as increased sensitivity, chromatographic efficiency, and speed, were recognized nearly 50 years ago. Over the past two decades, this approach has experienced rapid growth, driven by the emergence of mass spectrometry applications serving -omics sciences and the need for analyzing minute volumes of precious samples with ever higher sensitivity. While nanoscale liquid chromatography (flow rates <1 μL/min) has gained widespread recognition in proteomics, the adoption of microscale setups (flow rates ranging from 1 to 100 μL/min) for low molecular weight compound applications, including metabolomics, has been surprisingly slow, despite the inherent advantages of the approach. Highly heterogeneous matrices and chemical structures accompanied by a relative lack of options for both selective sample preparation and user-friendly equipment are usually reported as major hindrances. To facilitate the wider implementation of microscale analyses, we present here a comprehensive tutorial encompassing important theoretical and practical considerations. We provide fundamental principles in micro-chromatography and guide the reader through the main elements of a microflow workflow, from LC pumps to ionization devices. Finally, based on both our literature overview and experience, illustrated by some in-house data, we highlight the critical importance of the ionization source design and its careful optimization to achieve significant sensitivity improvement.

Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts

Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC–MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper’s surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.

Screening of Exosome-Derived Proteins and Their Potential as Biomarkers in Diagnostic and Prognostic for Pancreatic Cancer.

In the oncological area, pancreatic cancer is one of the most lethal diseases, with 5-year survival rising just 10% in high-development countries. This disease is genetically characterized by KRAS as a driven mutation followed by SMAD4, CDKN2, and TP53-associated mutations. In clinical aspects, pancreatic cancer presents unspecific clinical symptoms with the absence of screening and early plasmatic biomarker, being that CA19-9 is the unique plasmatic biomarker having specificity and sensitivity limitations. We analyzed the plasmatic exosome proteomic profile of 23 patients with pancreatic cancer and 10 healthy controls by using Nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS). The pancreatic cancer patients were subdivided into IPMN and PDAC. Our findings show 33, 34, and 7 differentially expressed proteins when comparing the IPMN vs. control, PDAC-No treatment vs. control, and PDAC-No treatment vs. IPMN groups, highlighting proteins of the complement system and coagulation, such as C3, APOB, and SERPINA. Additionally, PDAC with no treatment showed 11 differentially expressed proteins when compared to Folfirinox neoadjuvant therapy or Gemcitabine adjuvant therapy. So here, we found plasmatic exosome-derived differentially expressed proteins among cancer patients (IPMN, PDAC) when comparing with healthy controls, which could represent alternative biomarkers for diagnostic and prognostic evaluation, supporting further scientific and clinical studies on pancreatic cancer.

Proteomic Analysis of Dysfunctional Liver Sinusoidal Endothelial Cells Reveals Substantial Differences in Most Common Experimental Models of Chronic Liver Diseases.

Molecular markers of dedifferentiation of dysfunctional liver sinusoidal endothelial cells (LSEC) have not been fully elucidated. We aimed at deciphering the molecular profile of dysfunctional LSEC in different pathological scenarios. Flow cytometry was used to sort CD11b-/CD32b+ and CD11b-/CD32b- LSEC from three rat models of liver disease (bile duct ligation-BDL; inhaled carbon tetrachloride-CCl4; and high fat glucose/fructose diet-HFGFD). A full proteomic profile was performed applying nano-scale liquid chromatography tandem mass spectrometry (nLC-MS) and analyzed with PEAKS software. The percentage of CD32b- LSEC varied across groups, suggesting different capillarization processes. Both CD32+ and CD32b- LSEC from models are different from control LSEC, but differently expressed proteins in CD32b- LSEC are significantly higher. Heatmaps evidenced specific protein expression patterns for each model. Analysis of biological significance comparing dysfunctional CD32b- LSEC with specialized CD32b+ LSEC from controls showed central similarities represented by 45 common down-regulated proteins involved in the suppression of the endocytic machinery and 63 common up-regulated proteins associated with the actin-dependent cytoskeleton reorganization. In summary; substantial differences but also similarities in dysfunctional LSEC from the three most common models of liver disease were found, supporting the idea that LSEC may harbor different protein expression profiles according to the etiology or disease stage.

Enzymatic cocktail production by a co-cultivation Solid-State Fermentation for detergent formulation

The industry of detergents is one of the main claimers of enzymes. Here we show a promising technique, a co-cultivation Solid-State Fermentation (SSF) of Aspergillus niger and Aspergillus oryzae for the production of enzymatic cocktails. It was optimized by a central composite design where a proportion 75 % of wheat bran and 25 % of soybean husk as substrate and a conidia ratio 25 % A. niger and 75 % A. oryzae were the optimal conditions. Furthermore, the cocktail was analyzed by Nanoscale liquid chromatography coupled to tandem mass spectrometry (Nano LC-MS/MS) and it was concluded that 54.17 % of proteins belong to the enzymes of interest, where 28.79 % were amylases (308.47 IU/mL), 10.56 % were proteases (2228 IU/mL,), 13.47 % were cellulases (3.97 IU/mL) and 1.34 % were lipases (0.789 IU/mL). Finally, the enzymes presented a great stability in a wide range of pH and temperatures; and an excellent cleaning efficiency against cleaning control strips indicating that they could be used for formulations.

Application of nano-LC – nano-ESI – Orbitrap-MS for trace determination of four priority PFAS in food products considering recently established tolerable weekly intake (TWI) limits

An analytical method was developed and validated for the analysis of four priority perfluoroalkyl substances (PFAS), namely, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonate (PFOS) in food products using nanoscale liquid chromatography (nano-LC) coupled with nanoscale electrospray ionization (nano-ESI) and Orbitrap mass spectrometry (Orbitrap-MS) detection. The efficiency of two different nano-LC setups for chromatographic separation of selected PFAS was evaluated. The optimal LC separation of analytes was achieved using a reversed phase C18 (RP-C18) nano bore column with an integrated emitter. The effect of matrix concentration factor on signal suppression/enhancement was evaluated for different matrices. The method validation indicated analyte recoveries in the range 83–118% and within-laboratory reproducibility from 7 to 18%, while reanalysis of the materials from proficiency tests (PTs) showed that the accuracy of the obtained concentrations ranged from 85 to 124% of the provided consensus values. The method limits of quantification (m-LOQs) were set as first validation levels ranging from 0.001 to 0.3 ng g−1 sample depending on the type of the food group. The observed method performance characteristics met the criteria stated in Commission Regulation (EU) 2022/1428, Commission Recommendation (EU) 2022/1431, as well as Guidance Document on Analytical Parameters for the Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Food and Feed with regards to the compliance testing of PFAS maximum levels (MLs) and monitoring purposes. The elaborated method was applied for the analysis of selected priority PFAS in different food groups collected from the Latvian retail market.

Proteomic analysis of the mouse sperm acrosome - towards an understanding of an organelle with diverse functionality

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation.

P106 Identification of urine and serum diagnostic biomarkers of inflammatory bowel disease using a proteomic approach

Abstract Background Inflammatory bowel diseases (IBD) are chronic, heterogeneous, and inflammatory conditions mainly affecting the gastrointestinal tract. Currently, endoscopy is the gold standard test for assessing mucosal activity and healing in clinical practice; however, it is a costly, time-consuming, invasive, and uncomfortable procedure for patients. There is, therefore, a need for sensitive, specific, fast and non-invasive biomarkers for the diagnosis of IBD. Methods Label-free quantification by nanoscale liquid chromatography coupled to tandem mass spectrometry (nLC MS/MS) was performed to profile the urinary and serum proteomes of 100 patients with newly diagnosed IBD, before starting any treatment [50 patients with Crohn′s disease (CD) and 50 patients with ulcerative colitis (UC)] and 50 healthy controls (HC). Data Mining and Pattern Recognition techniques were used to identify hidden relationships that are not detectable by using the classical and most commonly used lineal classifiers; and thus, identifying potential markers able to discriminate the studied cohorts. Finally, we applied Ingenuity Pathway Analysis (IPA) to analyze the pathways and functions of differentially expressed proteins. Results Serum proteomics results revealed 45 differentially expressed proteins in the comparison between UC and HC groups, 32 proteins significantly expressed in CD versus HC, and 12 proteins in CD compared with UC. In urine samples, 110 proteins were significantly changed in UC versus HC, 50 proteins were differentially expressed between CD and HC, and a total of 31 proteins were significantly changed in CD compared with UC patients. Receiver operating characteristic curve (ROC) analysis found multiple proteins with high area under the curve values, up to 0.94, indicating that these serum and urine proteins are of value as new non-invasive diagnostic classifiers of IBD patients. For each study comparative, the 5 most significant classifiers were selected (Figures 1 and 2). IPA revealed multiple signaling pathways, including prothrombin activation, acute phase response signaling, complement and coagulation system and liver X receptor/retinoid X receptor (LXR/RXR) activation, altered in IBD patients compared to HC (p-value &amp;lt; 0.05) (Figures 3 and 4). Conclusion Our findings indicate that analysis of the urine and serum proteome using nLC MS/MS is a feasible approach for biomarker discovery. We identified several serum and urine proteins that could serve as new non-invasive markers for the diagnosis of IBD patients after further validation. After bioinformatic analyses, we found multiple proteins that may play important roles in the pathogenesis of IBD.

A bottom-up proteomics workflow for a system containing multiple organisms.

Discovery proteomics has been popularized to be essential in the investigator's biological toolbox. Many biological problems involve the interplay of multiple organisms. Herein, a bottom-up proteomics workflow was developed to study a system containing multiple organisms to promote a thorough understanding of how each interacts with the others. A label-free quantification proteomics workflow was developed with nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). This protocol describes a bottom-up proteomics workflow used to study differential protein expression in the context of fleas (Ctenocephalides felis felis) experimentally infected by the bacterium Bartonella henselae, the etiological agent of Cat Scratch Disease (CSD). Step-by-step instructions are provided for protein extraction, protein cleanup, total protein measurement, nanoLC-MS/MS data acquisition, and data analysis using Proteome Discoverer software. Comprehensive and exhaustive details are included to promote the adoption of this proteomics workflow in other laboratories. A proteomics protocol is detailed for a system containing multiple proteomes from different taxonomic lineages using CSD (cats bitten by fleas infected with Bartonella henselae) as a model. The operating protocol can be readily applied to other label-free proteomics work involving multiple proteomes from taxonomically distinct organisms.

Screening for Lipid-Metabolism-Related Genes and Identifying the Diagnostic Potential of ANGPTL6 for HBV-Related Early-Stage Hepatocellular Carcinoma.

Lipid metabolic reprogramming is one of the hallmarks of hepatocarcinogenesis and development. Therefore, lipid-metabolism-related genes may be used as potential biomarkers for hepatocellular carcinoma (HCC). This study aimed to screen for genes with dysregulated expression related to lipid metabolism in HCC and explored the clinical value of these genes. We screened differentially expressed proteins between tumorous and adjacent nontumorous tissues of hepatitis B virus (HBV)-related HCC patients using a Nanoscale Liquid Chromatography-Tandem Mass Spectrometry platform and combined it with transcriptomic data of lipid-metabolism-related genes from the GEO and HPA databases to identify dysregulated genes that may be involved in lipid metabolic processes. The potential clinical values of these genes were explored by bioinformatics online analysis tools (GEPIA, cBioPortal, SurvivalMeth, and TIMER). The expression levels of the secreted protein (angiopoietin-like protein 6, ANGPTL6) in serum were analyzed by ELISA. The ability of serum ANGPTL6 to diagnose early HCC was assessed by ROC curves. The results showed that serum ANGPTL6 could effectively differentiate between HBV-related early HCC patients with normal serum alpha-fetoprotein (AFP) levels and the noncancer group (healthy participants and chronic hepatitis B patients) (AUC = 0.717, 95% CI: from 0.614 to 0.805). Serum ANGPTL6 can be used as a potential second-line biomarker to supplement serum AFP in the early diagnosis of HBV-related HCC.

Proteomic Analysis Revealed Different Molecular Mechanisms of Response to PEG Stress in Drought-Sensitive and Drought-Resistant Sorghums.

Drought is the major limiting factor that directly or indirectly inhibits the growth and reduces the productivity of sorghum (Sorghum bicolor (L.) Moench). As the main vegetative organ of sorghum, the response mechanism of the leaf to drought stress at the proteomic level has not been clarified. In the present study, nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) technology was used to compare the changes in the protein expression profile of the leaves of drought-sensitive (S4 and S4-1) and drought-resistant (T33 and T14) sorghum varieties at the seedling stage under 25% PEG-6000 treatment for 24 h. A total of 3927 proteins were accurately quantitated and 46, 36, 35, and 102 differentially abundant proteins (DAPs) were obtained in the S4, S4-1, T14, and T33 varieties, respectively. Four proteins were randomly selected for parallel reaction monitoring (PRM) assays, and the results verified the reliability of the mass spectrometry (MS) results. The response mechanism of the drought-sensitive sorghum leaves to drought was attributed to the upregulation of proteins involved in the tyrosine metabolism pathway with defense functions. Drought-resistant sorghum leaves respond to drought by promoting the TCA cycle, enhancing sphingolipid biosynthesis, interfering with triterpenoid metabolite synthesis, and influencing aminoacyl-tRNA biosynthesis. The 17 screened important candidate proteins related to drought stress were verified by quantitative real-time PCR (qRT-PCR), the results of which were consistent with the results of the proteomic analysis. This study lays the foundation for revealing the drought-resistance mechanism of sorghum at the protein level. These findings will help us cultivate and improve new drought-resistant sorghum varieties.

Variations in Blood Platelet Proteome and Transcriptome Revealed Altered Expression of Transgelin-2 in Acute Coronary Syndrome Patients.

Proteomic analyses based on mass spectrometry provide a powerful tool for the simultaneous identification of proteins and their signatures. Disorders detection at the molecular level delivers an immense impact for a better understanding of the pathogenesis and etiology of various diseases. Acute coronary syndrome (ACS) refers to a group of heart diseases generally associated with rupture of an atherosclerotic plaque and partial or complete thrombotic obstruction of the blood flow in the infarct-related coronary artery. The essential role in the pathogenesis of ACS is related to the abnormal, pathological activation of blood platelets. The multifactorial and complex character of ACS indicates the need to explain the molecular mechanisms responsible for thrombosis. In our study, we performed screening and comparative analysis of platelet proteome from ACS patients and healthy donors. Two-dimensional fluorescence difference gel electrophoresis and nanoscale liquid chromatography coupled to tandem mass spectrometry showed altered expressions of six proteins (i.e., vinculin, transgelin-2, fibrinogen β and γ chains, apolipoprotein a1, and tubulin β), with the overlapping increased expression at the mRNA level for transgelin-2. Dysregulation in protein expression identified in our study may be associated with an increased risk of thrombotic events, correlated with a higher aggregability of blood platelets and induced shape change, thus explaining the phenomenon of the hyperreactivity of blood platelets in ACS.

Identification of Endogenous Peptides in Nasal Swab Transport Media used in MALDI-TOF-MS Based COVID-19 Screening

Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)–time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes proteinagonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthaseas a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Proteomics and UHPLC-DAD-Q/Orbitrap-MS used to identify impurities in andrographolide.

Andrographolide active pharmaceutical ingredient (API) is a semidiurnal diterpene lactone with significant antipyretic, antiviral, anti-inflammatory and anticancer activities. A large amount of andrographolide API could only be obtained by extraction from Andrographis paniculata. Therefore, there may be related compounds, plant proteins and other impurities in andrographolide API. In order to improve the safety of andrographolide related preparations, it was necessary to clarify the impurities and improve the quality standard of andrographolide API. The related compounds were identified by ultrahigh-performance liquid chromatography with diode-array detector quadrupole Orbitrap mass spectrometry (UHPLC-DAD-Q/Orbitrap-MS), and the residual proteins were determined by ultrafiltration combined with proteomics. The proteomics method included protein extraction, content determination, digestion, desalination and nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) analysis. Then, MS results were compared with Andrographis paniculata protein database by Peaks Studio. The results showed that 32 related compounds were putatively identified, of which 30 impurities were identified for the first time. Seven residual proteins together with 11 highly suspected proteins were uniquely identified, including the T1UNN5_ANDPA protein with the highest intensity. This study will provide useful information on the composition of andrographolide API, which is important for the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided on the necessity of ultrafiltration in the production process of related injections.

Recombinant Mosquito Densovirus with Bti Toxins Significantly Improves Pathogenicity against Aedes albopictus.

Mosquito densoviruses (MDVs) are mosquito-specific viruses that are recommended as mosquito bio-control agents. The MDV Aedes aegypti densovirus (AeDNV) is a good candidate for controlling mosquitoes. However, the slow activity restricts their widespread use for vector control. In this study, we introduced the Bacillus thuringiensis (Bti) toxin Cry11Aa domain II loop α8 and Cyt1Aa loop β6-αE peptides into the AeDNV genome to improve its mosquitocidal efficiency; protein expression was confirmed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Recombinant plasmids were transfected into mosquito C6/36 cell lines, and the expression of specific peptides was detected through RT-PCR. A toxicity bioassay against the first instar Aedes albopictus larvae revealed that the pathogenic activity of recombinant AeDNV was significantly higher and faster than the wild-type (wt) viruses, and mortality increased in a dose-dependent manner. The recombinant viruses were genetically stable and displayed growth phenotype and virus proliferation ability, similar to wild-type AeDNV. Our novel results offer further insights by combining two mosquitocidal pathogens to improve viral toxicity for mosquito control.

Characterisation of Endogenous Peptides Present in Virgin Olive Oil.

The low molecular weight peptide composition of virgin olive oil (VOO) is mostly unknown. We aimed to investigate the composition of the endogenous peptides present in VOO, the protein sources from which those peptides originate and their biological activities. A water-soluble extract containing peptides was obtained from VOO. The peptides were separated by size-exclusion using fast protein liquid chromatography, and the low molecular weight fraction (1600–700 kDa) was analysed by nanoscale liquid chromatography Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Nineteen new peptides were identified by Peaks database algorithm, using the available Olea europaea (cv. Farga) genome database. Eight new peptides were also identified by Peaks de novo sequencing. The protein sources of the peptides detected in the database by Peaks DB were identified by BLAST-P search. Seed storage proteins were among the most frequent sources of VOO peptides. BIOPEP software was used to predict the biological activities of peptides and to simulate (in silico) the proteolytic activity of digestive enzymes on the detected peptide sequences. A selection of synthetic peptides was obtained for investigation of their bioactivities. Peptides VCGEAFGKA, NALLCSNS, CPANGFY, CCYSVY and DCHYFL possessed strong ACE-inhibitory and antioxidant activities in vitro. Antioxidant peptides could play a role in VOO quality.

Differential Proteomic Analysis of Complex Mixtures by Label-Free nLC MS/MS.

After over two decades of constant evolution, proteomics can be truly considered nowadays as a high-throughput technique. Latest advances performed in sample preparation, instrumentation, and data analysis tools enable proteome-wide detection and quantification of proteins in complex samples.Label-free quantification by nanoscale liquid chromatography coupled online to tandem mass spectrometry (nLC MS /MS ) is a straightforward procedure for relative protein quantification. This approach allows to get deeper insights of what molecular changes are involved in the biological system we want to study in an unbiased manner.This chapter describes methods for sample preparation prior to mass spectrometry analysis. Besides, we describe a standard acquisition method, and some common bioinformatics analyses that help extracting biologically relevant information out of the achieved data.

Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation.

Background & AimsNADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process.MethodsThe NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed.ResultsWe identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid–induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage.ConclusionsThe ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.