Imidazolium labelling permits the sensitive mass-spectrometric detection of N-glycosides directly from serum.
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ABSTRACT:
SUBMITTER: Zhang YY
PROVIDER: S-EPMC8280963 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundGABAA receptor (GABAAR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I2 of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABAAR are deficient.Methodology/principal findingsThe aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABAAR. We have developed a new analytical tool for in vitro detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABAAR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABAAR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABAAR.Conclusion/significanceOur results provide new insights on the regulation of GABAAR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed.
| S-EPMC4072668 | biostudies-literature
Project description:Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 μg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
| S-EPMC3975687 | biostudies-literature
Project description:The detection and quantification of nanoplastics in aquatic environments is one of the major challenges in environmental and analytical research nowadays. The use of common analytical techniques for this purpose is not only hampered by the size of nanoplastics, but also because they are mainly made of carbon. In addition, the expected concentrations in environmental samples are below the detection limit of the majority of analytical techniques. In this context, the great detection capabilities of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in its Single Particle mode (SP-ICP-MS) have made of this technique a good candidate for the analysis of nanoplastics. Since the monitoring of carbon by ICP-MS faces several difficulties, the use of metal tags, taking advantage of the great potential of nanoplastics to adsorb chemical compounds, has been proposed as an alternative. In this perspectives paper, three different strategies for the analysis of polystyrene (PS) nanoplastics by SP-ICP-MS based on the use of metals species (ions, hydrophobic organometallic compound, and nanoparticles) as tags are presented and discussed. Advantages and disadvantages of each strategy, which rely on the labelling process, are highlighted. The metal nanoparticles labelling strategy is shown as a promising tool for the detection and quantification of nanoplastics in aqueous matrices by SP-ICP-MS.
| S-EPMC8659233 | biostudies-literature
Project description:Changes of glycosylation pattern in serum proteins have been linked to various diseases including cancer, suggesting possible development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between P value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multibiomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80~90% sensitivity, and 70~83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81~84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74% in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.
| S-EPMC3932261 | biostudies-literature
Project description:Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification from clinical samples that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 non-cancerous and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across non-cancerous and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to non-cancerous patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for non-cancerous) (p value < 0.001). Our results represent the first application of the alkylated hepcidin assay to clinical samples and demonstrate that the developed assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.
| S-EPMC6513854 | biostudies-literature
Project description:Virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. In the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. In this work, dengue virus was used as a model to test a new approach of combining broadly sensitive RT-PCR amplification of nearly any virus strain with subsequent serotype- and finer-level identification by mass spectrometry. PCR primers were appended with promoter sequences, such that the resulting PCR products could be transcribed into RNA. RNA fragments generated by guanosine-specific RNase T(1) digestion were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Viral serotypes were identified by comparing the pattern of observed fragment masses to a mass database. The database was created by computationally fragmenting 2517 dengue strains after each guanosine residue using the same primers. Computationally, all 2517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product. The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones.
| S-EPMC3391419 | biostudies-literature
Project description:Background:Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods:Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results:The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn's disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions:This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.
| S-EPMC6585069 | biostudies-literature
Project description:The reliability and information content of diethylpyrocarbonate (DEPC) as a covalent probe of protein surface structure has been improved when used appropriately with mass spectrometric detection. Using myoglobin, cytochrome c, and beta-2-microglobulin as model protein systems, we demonstrate for the first time that DEPC can modify Ser and Thr residues in addition to His and Tyr residues. This result expands the capability of DEPC as a structural probe because about 25% of the sequence of the average protein can now be covered using this covalent labeling reagent. In addition, we establish a new approach based on mass spectrometry to ensure the structural integrity of proteins during amino acid-specific covalent labeling reactions. This approach involves monitoring the extent of modification as a function of reagent concentration and allows any small-scale or local perturbations caused by the covalent label to be readily identified and avoided. Results indicate that these dose-response plots are much more reliable and generally applicable probes of possible protein structural changes than fluorescence or circular dichroism spectroscopies. These dose-response plots also provide a means of quantitatively comparing the reactivity of each modified residue. On the basis of comparisons to known X-ray crystal structures, we find that the solvent accessibility of the reactive atom in the side chain and the presence of a nearby charged residue most affect modification rates. Finally, this improved surface mapping method has been used to determine the effect of Cu(II) binding on the structure of beta-2-microglobulin. Results confirm that Cu(II) binds His31, but not any of the other three His residues, and changes the solvent accessibility of residues near His31 and near the N-terminus.
| S-EPMC4232213 | biostudies-literature
Project description:Neutrophils are the most abundant white blood cells in humans and play a vital role in several aspects of the immune response. Numerous reports have implicated neutrophil glycosylation as an important factor in mediating these interactions. We report here the application of high sensitivity glycomics methodologies, including matrix assisted laser desorption ionisation (MALDI-TOF) and MALDI-TOF/TOF analyses, to the structural analysis of N- and O-linked carbohydrates released from two samples of neutrophils, prepared by two separate and geographically remote laboratories. The data produced demonstrates that the cells display a diverse range of sialylated and fucosylated complex glycans, with a high level of similarity between the two preparations.
| S-EPMC2791480 | biostudies-literature
Project description:1. The structures of the alpha(2)- and alpha(3)-azopigments, prepared by diazotization of dog bile with ethyl anthranilate, were shown by mass spectrometry and g.l.c. to correspond to azobilirubin beta-d-xylopyranoside and azobilirubin beta-d-glucopyranoside respectively. 2. Both azopigments consist of a mixture of two methyl vinyl isomers having structures (IIIa) and (IIIb) for the alpha(2)-azopigment and structures (IVa) and (IVb) for the alpha(3)-azopigment. Separation of methyl vinyl isomers was obtained by t.l.c. or column chromatography performed on the acetylated azopigments. Hydrolysis of the less polar acetates derived from components (IIIa) and (IVa) gave rise to the azopigment (Ia), whereas hydrolysis of the more polar acetates derived from components (IIIb) and (IVb) gave rise to the azopigment acid (Ib). The positions of methyl and vinyl substituents in compounds (Ia) and (Ib) were assigned on the basis of their n.m.r. spectra. 3. Molecular ions in the mass spectra of the trimethylsilyl and acetyl derivatives of the azopigments indicated the presence of a pentose and a hexose conjugating sugar. 4. The ester functions linking the sugars to the propionic acid side chain of azobilirubin were demonstrated by ammonolysis and identification of the amide of azobilirubin as the aglycone derivative. 5. The sugar moieties were shown to occur as xylopyranose (alpha(2)) and glucopyranose (alpha(3)), bound at C-1, by application of a sequence of reactions performed on a micro-scale. The sugar hydroxyl groups were acetylated and the 1-acyl aglycone removed selectively by treatment with hydrogen bromide in acetic acid. Hydrolysis of the 1-bromo sugar acetates followed by acetylation afforded the alpha- and beta-xylopyranose tetra-acetates and alpha- and beta-glucopyranose penta-acetates, identified by a combination of g.l.c. and mass spectrometry. 6. The validity of this degradation scheme was confirmed (a) by g.l.c.-mass spectrometry identification of the alpha- and beta-1-propionyl derivatives of glucopyranose tetra-acetate, obtained from the alpha(3)-azopigment after final reaction with propionic anhydride; (b) by subjecting the acetates of alphabeta-glucopyranose, alphabeta-xylofuranose and alphabeta-glucofuranose to the same sequence of reactions.
| S-EPMC1178186 | biostudies-other