Dataset Information

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

ABSTRACT: 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)".

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michael Kreutzer

LAB HEAD: Prof. Dr. Michael O. Glocker

PROVIDER: PXD031150 | Pride | 2022-08-15

REPOSITORIES: Pride

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Publications

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Scherf Maximilian M Danquah Bright D BD Koy Cornelia C Lorenz Peter P Steinbeck Felix F Neamtu Andrei A Thiesen Hans-Jürgen HJ Glocker Michael O MO

Chembiochem : a European journal of chemical biology 20220912 20

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated ...[more]

PMID: 35950614

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Project description:Non-shivering thermogenesis in adipocytes is mediated by brown adipose tissue, purportedly through the sole action of uncoupling protein 1 (UCP1). The physiological relevance of UCP1-dependent thermogenesis has primarily been inferred from the attenuation of thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue beyond UCP1, such as reduced electron transport chain abundance. We show here that these secondary changes also encompass reduced expression of genes regulating fuel liberation, changes that would attenuate the capacity of any thermogenic pathway. Therefore, the quantitative contribution of UCP1-dependent and -independent thermogenesis is not fully understood. To mitigate the potentially confounding ancillary changes to brown adipose tissue of germline Ucp1-/- mice, we constructed mice with inducible adipocyte-selective disruption of Ucp1. We find that, while germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase B (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold-intolerance, indicative of a negative genetic interaction and thus a parallel thermogenic function. We find no evidence for impairments in insulation or non-shivering thermogenesis in skeletal muscle that would drive this phenotype. Furthermore, following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature adipocytes that coordinately restore UCP1 and CKB to brown adipose tissue, providing further evidence of their parallel thermogenic relationship. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy – through UCP1 and CKB – to promote cold-induced energy dissipation.

Dataset Information

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

Publications

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Similar Datasets

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