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Mass spectrometric ITEM-TWO analyses of interactions between human cardiac Troponin T (hcTnT) peptides and the M7 or the M11.7 antiTnT antibodies.

ABSTRACT: Molecular characterization of antibodies M7, and M11.7, as well as the antiTNF alpha antibody in addition to the synthetic peptides P11-P17 and P21-P32. The M7 and M11.7 anti-human cardiac Troponin T (antihcTnT) antibodies from Roche Diagnostics Deutschland GmbH (Mannheim, Germany) and the antiTNF alpha antibody from Thermo Fisher Scientific (Waltham, MA, USA, cat.no. 28401) were reconstituted and diluted with 200 mM ammonium acetate, pH 6.7, for nano-electrospray mass spectrometry. Molecular masses were determined. Synthesized peptides P11-P17 and P21-P32, delivered by 'Peptide&Elephants' (Henningsdorf, Germany), were dissolved in 200 mM ammonium acetate, pH 6.7, and also forwarded to nano-electrospray mass spectrometric analysis. Molecular masses and amino acid sequences were determined by mass spectrometric fragmentation of precursor ions. ITEM TWO: The antibodies M7, M11.7 and antiTNF alpha were incubated with selected synthesized peptides to generate solutions which contained the antibody and one synthesized peptide, dissolved in 200 mM ammonium acetate, pH 6.7. Antibody – peptide solutions were subjected to nano electrospray mass spectrometric ITEM-TWO analyses to investigate the binding behavior of the antibody and synthesized peptides, such as the apparent binding energies and dissociation constants of the complex dissociation reactions in the gas phase.

INSTRUMENT(S): Synapt G2-S HDMS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michael Kreutzer

LAB HEAD: Michael O. Glocker

PROVIDER: PXD058812 | Pride | 2025-05-20

REPOSITORIES: Pride

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Project description:Background: Most asthmatic patients have high serum levels of IgE directed against common environmental allergens such as house dust mite, animal danders and moulds. However the presence of specific IgE against individual allergens alone does not account for asthma. Many individuals are atopic but not asthmatic. Precise knowledge of the serum IgE specificity repertoire in asthmatic and non-asthmatic patients would substantially help in understanding the pathogenesis of the disease and at the same time facilitate the treatment and the implementation of preventive measures. Methods: We developed a microarray immunoassay containing 103 common allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals from families whose members had a documented history of asthma and atopy. The results were analyzed using k-means clustering to investigate whether IgE reactivity profiles correlated with asthma, disease severity and age of onset as well as with other atopic conditions such as rhinitis, conjunctivitis and eczema. We trained an artificial neural network (ANN) using the serum reactivity data to identify individuals as asthmatic and non-asthmatic. Results: Individual sera showed clear differences in the number and the combinations of allergens recognized as well as in the level of specific IgE. While the presence of specific IgE against single allergens correlated poorly with the pathological conditions examined k-means clustering analysis unraveled that a particular profile was significantly associated with asthma (p <1E-8). An ANN-based algorithm, calibrated with the profile reactivity data correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Conclusions: Our analysis demonstrates that asthma may be a higher-order phenomenon related to patterns of response and not attributable to single antibody reactions. This information sheds new light on the risk of developing the disease and can be readily utilized in combination with an ANN-based tool to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile. The study consisted of a total of 872 sera samples: 485 individuals were diagnosed with asthma, 342 were classified as non asthmatic, a remaining 45 were classified as unconfirmed asthma diagnosis. The serum IgE reactivity profiles were analyzed against 103 allergens representative of 11 distinct allergen classes. Quantification of bound IgE: The fluorescence signal was acquired using ProScanArray Express™ version 3.0 software. PMC reading values of individual spots were corrected against the internal negative control to identify signals above background. Duplicate measurements of individual allergens were utilized. The signal collected from the allergens was interpolated with an external calibration curve to obtain the IU/ml value (see supplementary file GSE20020_IU_ml values.txt), and translated into a Class Score by plotting the data in a standard reactivity scale (see Sample data tables). Class Score values: (CLASS 0 (less than 0.35 IU/ml); CLASS 1 (0.35-0.7 IU/ml); CLASS 2 (0.71-3.5 IU/ml); CLASS 3 (3.51-17.5 IU/ml); CLASS 4 (17.51-50 IU/ml); CLASS 5 (50.01-100 IU/ml).

Project description:The cost of Carica papaya production through seed-based propagation is increased by sex segregation, making in vitro techniques a more appealing option for clonal propagation. Inducing embryogenic callus with 2,4-dichlorophenoxyacetic acid (2,4-D) hold the potential to large-scale cloning, although the molecular mechanisms underlying this process are still not well understood. In this study, we performed a temporal analysis in the proteome of C. papaya callus to identify the key players involved in embryogenic differentiation. Mature zygotic embryos were used as explants and treated with 20 μM 2,4-D to induce embryogenic callus. Total proteins were extracted at 0, 7, 14, and 21 days (T0, T7, T14, and T21), and 1407 proteins were identified using bottom-up proteomic approach. Comparative proteomics revealed 957 differentially accumulated proteins (DAPs) (p<0.05 and log2FC >0.585 or <-0.585) in at least one comparison between the analyzed induction times points. The clustering analysis revealed four clusters with distinct patterns of protein accumulation throughout the embryogenic callus induction treatment. The cluster 1 contains 386 DAPs that accumulated at all analyzed times after treatment with 2,4-D. In contrast, cluster 2 contains 165 DAPs that decrease in abundance during the induction. The cluster 3 contains 251 proteins that are most abundant just after the start of incubation in 2,4-D (T7) and cluster 4 grouped 155 proteins that accumulate after callus formation. Functional analysis revealed that proteins involved with reserve storage and seed maturation were more abundant in the explant at T0 and decreased as callus formation progressed. Biological processes involving carbohydrate and amino acid metabolism, aerobic respiration, and protein catabolic processes were enriched after induction treatment. Regulatory proteins, including histone deacetylase (HDT3) and argonaute 1, were more abundant after the start of induction treatment with 2,4-D, suggesting their role in acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14.3.3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation, hormone response, and SAM metabolism. Our findings emphasize the modulation of the proteome at different stages during embryogenic callus initiation and identify regulatory proteins that might be involved with the activation of this process.

			Action	DRS
	DB_PCR_pep_20221117.fasta	Fasta
	F417274.dat	Other
	F417274.mgf	Mgf
	F417275.dat	Other
	F417275.mgf	Mgf

Dataset Information

Mass spectrometric ITEM-TWO analyses of interactions between human cardiac Troponin T (hcTnT) peptides and the M7 or the M11.7 antiTnT antibodies.

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