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Characterization of Phosphorylation-dependent Antibody Binding to Cancer-mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping - Thermodynamic Weak-force Order Analysis.

ABSTRACT: We describe mass spectrometric antibody-peptide binding motif analyses of a monoclonal antibody (clone 18E9.D9, BioLegend, London, England) from mouse which was raised against the predominantly occuring phosphorylated linker sequence HpTGEKP of human C2H2 zinc finger proteins. As the anti-HpTGEKP antibody is assumed to bind to related phosphorylated C2H2 ZNF protein linker sequences as well, specific amino acid exchanges were investigated for evaluating their importance for immune complex formation. Two point mutations of the zinc finger protein 331 (ZNF331) gene, causing single amino acid exchanges R9I and H11Y, respectively, have been found to be significantly enriched in Uterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma. Since the H11Y amino acid exchange affects histidine residue H1 of the HpTGEKP linker motif which is recognized by the anti-HpTGEKP antibody, we examined in this work whether the in several cancers mutated linker amino acid sequence YTGEKP and its phosphorylated derivatives YpTGEKP, pYTGEKP, and pYpTGEKP would be bound by the anti-HpTGEKP antibody. Immune complex formation ability was compared to that of peptides HTGEKP, HpTGEKP, and HpTGKKP, respectively, which served as controls. Binding behaviors towards the anti-HpTGEKP antibody were investigated by "Intact Transmission Epitope Mapping - Thermodynamic Weak-force Order (ITEM-TWO)".

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michael Kreutzer

LAB HEAD: Prof. Dr. Michael O. Glocker

PROVIDER: PXD035818 | Pride | 2023-01-25

REPOSITORIES: Pride

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Epitope fine mapping by mass spectrometry: Investigations of immune complexes consisting of monoclonal anti-HpTGEKP antibody and zinc finger protein linker phospho-hexapeptides.

Project description:We describe the mass spectrometric analysis of the binding strength of a monoclonal mouse antibody (clone 18E9.D9, BioLegend, London, England) which was raised against the predominantly occuring phosphorylated linker sequence, HpTGEKP, of C2H2 zinc finger proteins (ZNF proteins) using a synthetic phospho-hexapeptide as epitope peptide. The epitope peptide resembles the sequence motive which occurs with highest frequency in all C2H2 zinc finger proteins. As this anti-HpTGEKP antibody is assumed to bind to various related phosphorylated C2H2 ZNF protein linker sequences as well, related phospho-peptide sequences were investigated for binding strength differences. To select the top ten phosphorylated linker sequence motives of all human C2H2 ZNF proteins, all potential C2H2 ZNF gene linker sequences were extracted from the Human Genome Reference Consortium database (Build 38, patch release 13). DNA sequence stretches with stop-codons were removed and the DNA codons from the remaining sequences were translated. The resulting amino acid sequences of all C2H2 ZNF protein linkers were ranked according to their frequencies of occurrence. From these top ten C2H2 ZNF protein linker sequences the respective phospho-hexapeptides were synthesized and their binding behaviors towards the anti-HpTGEKP phospho-peptide antibody were investigated by "Intact Transmission Epitope Mapping – Thermodynamic Weak-force Order (ITEM-TWO)".

2022-08-15 | PXD031150 | Pride

Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization

Project description:We describe theoretical concepts and explain practical procedures of an electrospray mass spectrometry method, termed “Intact Transmission Epitope Mapping – Thermodynamic Weak-force Order (ITEM-TWO)”, by which can be determined apparent binding energies and dissociation constants of immune complex dissociation reactions in the gas phase. The protocol was developed using natural protein-ligand complexes (myoglobin and RNAse S) and was tested with two immune complexes (FLAG peptide - antiFLAG antibody and Troponin I epitope peptide - anti-Troponin I antibody). ITEM-TWO is a rapid method which requires very little sample consumption for identification of protein bound ligands and for determination of the gas phase protein-ligand complex binding strength.

2020-10-29 | PXD021296 | Pride

Intact Transition Epitope Mapping-Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)

Project description:ITEM-THREE is a novel mass spectrometry-based epitope mapping method. To perform ITEM-THREE, antibody-epitope peptide immune-complexes are first generated in electrospray-compatible solutions in which the antibodies maintain their in-solution functionality, by adding an antibody of interest to a mixture of peptides/ digest of antigen from which at least one of the peptides holds the antibody's epitope. This mixture is directly nano-electrosprayed without purification. Identification of the epitope peptide is executed within a mass spectrometer which provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a step-wise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope containing peptide fragment ions are recorded and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen.

2019-06-03 | PXD013823 | Pride

Intact Transition Epitope Mapping- Thermodynamic Weak-force Order (ITEM-TWO)

Project description:Most important characteristics of antibodies are that they typically strongly bind to specific epitopes, thereby expressing low dissociation constants (KDs) and high Gibbs free binding energies (delta Gs). In this study, we describe an off-line nano-electrospray mass spectrometry method, termed ITEM-TWO, which enables one to simultaneously identify epitopes and obtain characteristic gas phase dissociation constants of antibody - epitope interactions in a single experiment. To validate the method, we characterize the interaction between an antiHistag antibody and its epitope peptide obtained from a tryptic digest of a His-tag containing beta actin. In a mixture of solution 1 (tryptic digest of a His-tag containing recombinant human beta-actin protein in 30 mM ammonium bicarbonate) and solution 2 (antiHis-tag antibody in 200 mM ammonium acetate) the specific immune complexes form (molar ratio 2.2 : 1). Without any purification steps this mixture (solution 3) is then electrosprayed. With the aid of a quadrupole ion filter, the immune complex ions are separated from unbound peptide ions. Increasing the voltage difference (DeltaCV) in the subsequent collision cell results in collision induced dissociation (CID) by which the epitope peptide(s) is(are) released from the immune complex. The mass(es) of the complex-released peptide(s) is(are) then measured in a ToF analyzer by which the epitope is identified. A step-wise increase in DeltaCV allows the simultaneous determination of the intensities of (i) the surviving ionized immune complexes together with (ii) released epitope peptide ions plus (iii) the left behind antibody ions. From the ions´ normalized intensity ratios are deduced the apparent gas phase dissociation constants (KD#m0g) and the apparent dissociation energies over temperature values (delta G#m0g / T) of the gas phase dissociation processes.

2019-11-07 | PXD016024 | Pride

Intact Transition Epitope Mapping - Force differences between Original and Unusual Residues (ITEM-FOUR)

Project description:Human cardiac Troponin I epitope peptides were incubated with monoclonal anti-cardiac Troponin I antibody. Immune complexes were subjected to nano electrospray mass spectrometry in order to determine apparent binding energies and dissociation constants of immune complex dissociation reactions in the gas phase.

2023-01-17 | PXD038365 | Pride

Epitope mapping of an anti-plasmodium pfMSP119 antibody by Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes

Project description:Rapid diagnostic tests are first line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population screening assays, high quality reagents and well characterized antigens and antibodies are needed. An important property of antigen - antibody binding is recognition specificity which best can be estimated by mapping an antibodys epitope. The MBP-pfMSP119 antigen was chosen as mutual target for population screening. Also, since an anti-pfMSP119 antibody is planned to function as positive control in screening assays, its binding characteristics to the antigen was investigated. Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE) was carried out to map the epitope of a monoclonal anti-pfMSP119 antibody, i.e. the recognized area on the MBP-pfMSP119 antigen surface. The MBP-pfMSP119 fusion protein was cloned and expressed in E.coli which then was enriched by affinity purification on amylose resin. The enriched and purified MBP-pfMSP119 fusion protein was structurally and functionally characterized before and after high pressure-assisted tryptic digestion or after GluC digestion of the reduced and alkylated fusion protein.

2020-08-24 | PXD019717 | Pride

Pre-existing cross-reactive antibody responses do not significantly impact inactivated COVID-19 vaccine-induced neutralization

Project description:Recent exposure to seasonal coronaviruses (sCoVs) may stimulate cross-reactive antibody responses against SARS-CoV-2. Previous studies have shown divergent results regarding protective or damaging immunity induced by prior exposure to sCoVs. It is still unknown whether pre-existing humoral immunity may play a role in the vaccine-induced neutralization and antibody responses. In this study, we collected 36 paired sera in healthy volunteers before and after immunization with inactivated SARS-CoV-2 vaccines, and analyzed the distribution and intensity of pre-existing antibody responses at the epitope level before vaccine immunization, as well as the relationship between pre-existing sCoVs immunity and vaccine-induced neutralization.

2021-10-13 | E-MTAB-11084 | biostudies-arrayexpress

Pre-existing cross-reactive antibody responses do not significantly impact inactivated COVID-19 vaccine-induced neutralization

2021-10-29 | E-MTAB-11117 | biostudies-arrayexpress

A combined gas phase separation strategy for ADP-ribosylated peptides

Project description:ADP-ribosylation (ADPr) is a posttranslational modification that is best studied using mass spectrometry. Method developments that are permissive to low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation/separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants, and the peptide inputs for ADPr peptide enrichment. At increasingly higher compensation voltages, ADPr peptides were more stable whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings, provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined gas phase separation strategy is a valuable addition to any ADP-ribosylome workflow.

2023-08-30 | PXD040898 | Pride

Glomerular Transcriptome from European Renal cDNA Bank subjects and living donors

Project description:Glomerular Transcriptome from European Renal cDNA Bank subjects and living donors. Samples included in this analysis have been previously analyzed using older CDF definitions and are included under previous GEO submissions - GSE47183 (chronic kidney disease samples), and GSE32591 (IgA nephropathy samples). RNA from the glomerular compartment of was extracted and processed for hybridization on Affymetrix microarrays, annotated using Human Entrez Gene ID custom CDF version 19. This dataset is part of the TransQST collection.

2018-01-24 | E-GEOD-104948 | biostudies-arrayexpress

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