Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein.

A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

Novel LC- ESI-MS/MS method for desvenlafaxine estimation human plasma: application to pharmacokinetic study.

A simple, sensitive and specific liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the quantification of desvenlafaxine in human plasma using desvenlafaxine d6 as an internal standard (IS). Chromatographic separation was performed using a Thermo-BDS hypersil C8 column (50 × 4.6 mm, 3 µm) with an isocratic mobile phase composed of 5 mM ammonium acetate buffer: methanol (20:80, v/v), at a flow rate of 0.80 mL/min. Desvenlafaxine and desvenlafaxine d6 were detected with proton adducts at m/z 264.2/58.1 and 270.2/ 64.1 in multiple reaction monitoring positive mode, respectively. Liquid-liquid extraction was used to extract the drug and the IS. The method was linear over the concentration range 1.001-400.352 ng/mL with a correlation coefficient of ≥0.9994. This method demonstrated intra and inter-day precision within 0.7-5.5 and 1.9-6.8%, and accuracy within 95.3-107.4 and 93.4-99.5%. Desvenlafaxine was found to be stable throughout the freeze-thaw cycles, bench-top and long-term matrix stability studies. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of desvenlafaxine in pharmaceutical dosage forms.

Interdisciplinary gene manipulation, molecular cloning, and recombinant expression of modified human growth hormone isoform-1 in E. coli system.

BACKGROUND: Growth hormone (GH) is a hormone that promotes growth, cell reproduction, and cell restoration in humans and animals. OBJECTIVES: Production of recombinant human growth hormone (rhGH) in Escherichia coli (E. coli) and assessment of its characteristics and proliferation stimulatory activity. METHODS: The hGH gene was cloned into a pET 3a expression vector and transformed into a competent E. coli cell. The refolded hGH was purified, Western blot and batch fermentation were performed. Cell cytotoxicity was tested on Vero cells, and MALDI-TOF and Nano-LC-ESI MS/MS were used for protein and target peptide analysis. RESULTS: Induced rhGH was purified with a concentration of 511.9 mg/ml. Western blot confirmed the molecular identity of rhGH, showing a single 22 kDa band. The bacterial growth at OD600 after 24 h in batch fermentation was 9.78 ± 0.26, and wet cell weight (WCWg/L) was 15.2 ± 0.32. Purified rhGH activity on Vero cells was 0.535 IU/mg. LC-MS/MS analysis revealed a score of 70.51 % and coverage of 60.37 %. CONCLUSION: Biologically active native rhGH protein was successfully expressed in the Prokaryotic system. Our goal is to increase its production on a pilot level in the native form at a high activity effect identical to isoform 1.

A robust nanoLC high-resolution mass spectrometry methodology for the comprehensive profiling of lactic acid bacteria in milk kefir.

Consumer attention to functional foods containing probiotics is growing because of their positive effects on human health. Kefir is a fermented milk beverage produced by bacteria and yeasts. Given the emerging kefir market, there is an increasing demand for new methodologies to certify product claims such as colony-forming units/g and bacterial taxa. MALDI-TOF MS proved to be useful for the detection/identification of bacteria in clinical diagnostics and agri-food applications. Recently, LC-MS/MS approaches have also been applied to the identification of proteins and proteotypic peptides of lactic acid bacteria in fermented food matrices. Here, we developed an innovative nanoLC-ESI-MS/MS-based methodology for profiling lactic acid bacteria in commercial and artisanal milk kefir products as well as in kefir grains at the genus, species and subspecies level. The proposed workflow enables the authentication of kefir label claims declaring the presence of probiotic starters. An overview of the composition of lactic acid bacteria was also obtained for unlabelled kefir highlighting, for the first time, the great potential of LC-MS/MS as a sensitive tool to assess the authenticity of fermented foods.

Mechanistic insights of Cucumis melo L. seeds for gastrointestinal muscle spasms through calcium signaling pathway-related gene regulation networks in WGCNA and in vitro, in vivo studies.

BACKGROUND: In addition to the nutritional benefits of Cucumis melo L., herbalists in Pakistan and India employ seeds to treat various ailments. This study aimed to determine the regulatory role of C. melo seeds in calcium-mediated smooth muscle contraction. METHODS: We identified and quantified the phytochemicals of C. melo with LC ESI-MS/MS and HPLC, then conducted in vitro and in vivo tests to confirm the involvement in smooth muscle relaxation. Then, diarrhea-predominant irritable bowel syndrome gene datasets from NCBI GEO were acquired, DEGs and WGCNA followed by functional enrichment analysis. Next, molecular docking of key genes was performed. RESULTS: The quantification of C. melo seeds revealed concentrations of rutin, kaempferol, and quercetin were 702.38 µg/g, 686.29 µg/g, and 658.41 µg/g, respectively. In vitro experiments revealed that C. melo seeds had a dose-dependent relaxant effect for potassium chloride (80 mM)-induced spastic contraction and exhibited calcium antagonistic response in calcium dose-response curves. In in vivo studies, Cm.EtOH exhibited antidiarrheal, antiperistaltic, and antisecretory effects. The functional enrichment of WGCNA and DEGs IBS-associated pathogenic genes, including those involved in calcium-mediated signaling, MAPK cascade, and inflammatory responses. MAPK1 and PIK3CG were identified as key genes with greater binding affinity with rutin, quercitrin, and kaempferol in molecular docking. CONCLUSIONS: The bronchodilator and antidiarrheal effects of C. melo were produced by altering the regulatory genes of calcium-mediated smooth contraction.

Metabolomics based mechanistic insights to vasorelaxant and cardioprotective effect of ethanolic extract of Citrullus lanatus (Thunb.) Matsum. & Nakai. seeds in isoproterenol induced myocardial infraction.

BACKGROUND: Cardiovascular diseases (CVDs) are a significant cause of morbidity and death in the current world, posing a challenge to both developing and industrialized nation's health systems. Citrullus lanatus (Thunb.) Matsum. & Nakai. seeds have long been utilized to supplement and enhance health and treat cardiovascular illnesses. However, its treatments for CVDs are still unknown. More research is required to fully comprehend the impact of C. lanatus seeds on vasorelaxation and myocardial infractions. PURPOSE: Therefore, an integrated metabolomics profiling technique was used to investigate possible pathways of C. lanatus in isoproterenol (ISO)-induced myocardial infarction (MI). Isoproterenol causes long-term cardiac hypertrophy by causing cardiomyocyte compensatory loss, eventually leading to heart failure. METHODS: In vitro models of vasoconstriction, atrium, and in vivo models of invasive blood pressure measurement and isoproterenol (ISO) induced cardiac hypertrophy in rats were used to understand underlying mechanistic by LC-MS/MS based dynamic metabolomics analysis of the serum and heart samples to be investigated the effect of ethanolic extract of C. lanatus (Cl.EtOH). RESULTS: Cl.EtOH exhibited vasorelaxant, negative chronotropic, and inotropic effects in in-vitro models whereas, a potent hypotensive effect was observed in normotensive rats. The Cl.EtOH protected the animals from ISO-induced myocardial infarction (MI) with therapeutic interventions in left ventricular thickness, cardiomyocyte hypertrophy, mRNA gene expression, biochemical assays, and metabolomic profiling of serum and heart tissues. CONCLUSIONS: For the first time, our study confirmed that C. lanatus seeds (Cl.EtOH) possess significant antihypertensive and prevent ISO-induced myocardial infarction. These findings comprehensively demonstrated mechanistic insights of Cl.EtOH in vasorelaxation and myocardial infarction. The current study provides evidence for further mechanistic studies and the development of C. lanatus seeds as a potential therapeutic intervention for patients with cardiovascular disorders.

Free low-molecular weight phenolics composition and bioactivity of Vaccinium padifolium Sm fruits.

Uveira-da-serra (Vaccinium padifolium Sm) is a native blueberry from Madeira Island (Portugal). In this study, the free low-molecular weight phenolic composition of Vaccinium padifolium berries (uva-da-serra - UdS), was established using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) strategy combined with liquid chromatography electrospray ionization tandem mass spectroscopy (LC-ESI-MSMS). Total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical-scavenging activities and oxygen radical absorbance capacity (ORAC) were also evaluated. Twenty-six phenolic compounds were identified in the UdS, being chlorogenic acid (17.4 mg/g DW), epigallocatechin (2.33 mg/g DW), caffeic acid (0.66 mg/g DW), quercetin-3-glucoside (0.38 mg/g DW) and myricetin (0.33 mg/g DW) the predominant compounds. As far we are aware, this is the first time that the free low molecular weight phenolic composition of Vaccinium padifolium Sm is characterized, also unveiling (-)epigallocatechin gallate, o-coumaric acid and m-coumaric acids presence in a Vaccinium specie. TPC (3021.8 mg GAE/100 g DW), TFC (2645.2 mg QE/100 g DW), DPPH (20509.0 µmol TE/g DW), ORAC (18510.0 µmol TE/g DW) and ABTS (19338.0 µmol TE/g DW) suggest a high antioxidant potential which is to health benefits including on cardiovascular and neurodegenerative disease prevention, making UdS a useful biosource with potential applications in food, pharmaceutical and cosmetic industries.

Mass spectrometry-based protein and peptide profiling for food frauds, traceability and authenticity assessment.

The ever-growing use of mass spectrometry (MS) methodologies in food authentication and traceability originates from their unrivalled specificity, accuracy and sensitivity. Such features are crucial for setting up analytical strategies for detecting food frauds and adulterations by monitoring selected components within food matrices. Among MS approaches, protein and peptide profiling has become increasingly consolidated. This review explores the current knowledge on recent MS techniques using protein and peptide biomarkers for assessing food traceability and authenticity, with a specific focus on their use for unmasking potential frauds and adulterations. We provide a survey of the current state-of-the-art instrumentation including the most reliable and sensitive acquisition modes highlighting advantages and limitations. Finally, we summarize the recent applications of MS to protein/peptide analyses in food matrices and examine their potential in ensuring the quality of agro-food products.

Analysis of Differentially Expressed Proteins Involved in Autoimmune Cirrhosis and Normal Serum by iTRAQ Proteomics.

PURPOSE: In order to study the candidate biomarkers in autoimmune cirrhosis (AIC). EXPERIMENTAL DESIGN: Isobaric tags are first implemented for relative and absolute quantitation technology on proteins prepared from serum obtained from AIC and normal controls. Proteins found to be differentially expressed are identified with liquid chromatography electrospray ionization tandem mass spectrometry by using a Q Exactive classic ion trap mass spectrometer. RESULTS: 108 proteins (32 upregulated and 76 downregulated proteins) are identified from AIC samples, compared with the normal controls. Gene Ontology enrichment analysis, KEGG pathway analysis, and protein-protein interaction map by STRING show that they associate with multiple functional groups, including ion binding activity, peptidase activity, and enzyme regulator activity. Finally, the von Willebrand factor, insulin-like growth factor-binding protein complex acid labile subunit, transthyretin, adiponectin proteins are identified with western blot as candidate biomarkers for AIC. CONCLUSIONS AND CLINICAL RELEVANCE: These findings offer a comprehensive profile of the AIC proteome about candidate biomarkers and provide a useful basis for further analysis of the pathogenic mechanism of AIC.

Reliable identification of lactic acid bacteria by targeted and untargeted high-resolution tandem mass spectrometry.

Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity. In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/MS for LAB identification at genus, species and sub-species level by using the sequence regions 33-52 and 72-82 of the S16 ribosomal protein as proteotypic peptide markers. The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Combined analysis of DNA methylation and cell cycle in cancer cells.

DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.