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

Overview

Journal Chembiochem

Specialty Biochemistry

Date 2022 Aug 11

PMID 35950614

Authors

Maximilian Scherf

Bright D Danquah

Cornelia Koy

Peter Lorenz

Felix Steinbeck

Andrei Neamtu

Hans-Jurgen Thiesen

Michael O Glocker

Affiliations

Soon will be listed here.

Abstract

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, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

Citing Articles

Mass spectrometry-complemented molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the circumsporozoite protein's C-terminal domain.

Opuni K, Russ M, Geens R, Vocht L, Wielendaele P, Debuy C Comput Struct Biotechnol J. 2024; 23:3300-3314.

PMID: 39296809 PMC: 11409006. DOI: 10.1016/j.csbj.2024.08.023.

Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer.

Rower C, Olaleye O, Bischoff R, Glocker M Biomolecules. 2024; 14(1).

PMID: 38254624 PMC: 10813730. DOI: 10.3390/biom14010024.

Intact Transition Epitope Mapping-Serological Inspection by Epitope EXtraction (ITEM-SIX).

Zammataro A, Koy C, Russ M, Rower C, Glocker M Molecules. 2023; 28(7).

PMID: 37049857 PMC: 10096252. DOI: 10.3390/molecules28073092.

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

Rower C, Ortmann C, Neamtu A, El-Kased R, Glocker M Biomolecules. 2023; 13(1).

PMID: 36671572 PMC: 9856199. DOI: 10.3390/biom13010187.

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

Scherf M, Danquah B, Koy C, Lorenz P, Steinbeck F, Neamtu A Chembiochem. 2022; 23(20):e202200390.

PMID: 35950614 PMC: 9826235. DOI: 10.1002/cbic.202200390.

References

Lew D, Kornbluth S . Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 1996; 8(6):795-804. DOI: 10.1016/s0955-0674(96)80080-9. View

Lustrek M, Lorenz P, Kreutzer M, Qian Z, Steinbeck F, Wu D . Epitope predictions indicate the presence of two distinct types of epitope-antibody-reactivities determined by epitope profiling of intravenous immunoglobulins. PLoS One. 2013; 8(11):e78605. PMC: 3823795. DOI: 10.1371/journal.pone.0078605. View

Saper C . A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem. 2008; 57(1):1-5. PMC: 2605712. DOI: 10.1369/jhc.2008.952770. View

Abbott W, Damschroder M, Lowe D . Current approaches to fine mapping of antigen-antibody interactions. Immunology. 2014; 142(4):526-35. PMC: 4107663. DOI: 10.1111/imm.12284. View

Suzuki K, Sako K, Akiyama K, Isoda M, Senoo C, Nakajo N . Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2. Sci Rep. 2015; 5:7929. PMC: 4300507. DOI: 10.1038/srep07929. View

Jen J, Wang Y . Zinc finger proteins in cancer progression. J Biomed Sci. 2016; 23(1):53. PMC: 4944467. DOI: 10.1186/s12929-016-0269-9. View

Luo L, Ando S, Sakamoto Y, Suzuki T, Takahashi H, Ishibashi N . The formation of perinucleolar bodies is important for normal leaf development and requires the zinc-finger DNA-binding motif in Arabidopsis ASYMMETRIC LEAVES2. Plant J. 2019; 101(5):1118-1134. PMC: 7155070. DOI: 10.1111/tpj.14579. View

Rould M, Nekludova L, Pabo C . Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure. 1996; 4(10):1171-80. DOI: 10.1016/s0969-2126(96)00125-6. View

Munro D, Ghersi D, Singh M . Two critical positions in zinc finger domains are heavily mutated in three human cancer types. PLoS Comput Biol. 2018; 14(6):e1006290. PMC: 6040777. DOI: 10.1371/journal.pcbi.1006290. View

10.

Nishi H, Shaytan A, Panchenko A . Physicochemical mechanisms of protein regulation by phosphorylation. Front Genet. 2014; 5:270. PMC: 4124799. DOI: 10.3389/fgene.2014.00270. View

11.

Weiss G, Watanabe C, Zhong A, Goddard A, Sidhu S . Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc Natl Acad Sci U S A. 2000; 97(16):8950-4. PMC: 16802. DOI: 10.1073/pnas.160252097. View

12.

Rower C, Vissers J, Koy C, Kipping M, Hecker M, Reimer T . Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue. Anal Bioanal Chem. 2009; 395(8):2443-56. DOI: 10.1007/s00216-009-3187-9. View

13.

Laity J, Lee B, Wright P . Zinc finger proteins: new insights into structural and functional diversity. Curr Opin Struct Biol. 2001; 11(1):39-46. DOI: 10.1016/s0959-440x(00)00167-6. View

14.

Lambert S, Jolma A, Campitelli L, Das P, Yin Y, Albu M . The Human Transcription Factors. Cell. 2018; 172(4):650-665. DOI: 10.1016/j.cell.2018.01.029. View

15.

Rizkallah R, Alexander K, Hurt M . Global mitotic phosphorylation of C2H2 zinc finger protein linker peptides. Cell Cycle. 2011; 10(19):3327-36. PMC: 3233627. DOI: 10.4161/cc.10.19.17619. View

16.

Opuni K, Koy C, Russ M, Reepmeyer M, Danquah B, Weresow M . ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the MSP1 antigen. J Biol Chem. 2020; 295(44):14987-14997. PMC: 7606685. DOI: 10.1074/jbc.RA120.014802. View

17.

Bannister D, Popovic B, Sridharan S, Giannotta F, Filee P, Yilmaz N . Epitope mapping and key amino acid identification of anti-CD22 immunotoxin CAT-8015 using hybrid β-lactamase display. Protein Eng Des Sel. 2010; 24(4):351-60. PMC: 3049344. DOI: 10.1093/protein/gzq114. View

18.

Danquah B, Yefremova Y, Opuni K, Rower C, Koy C, Glocker M . Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO). J Proteomics. 2019; 212:103572. DOI: 10.1016/j.jprot.2019.103572. View

19.

Danquah B, Opuni K, Roewer C, Koy C, Glocker M . Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization. Molecules. 2020; 25(20). PMC: 7587528. DOI: 10.3390/molecules25204776. View

20.

Hamed M, Siam R, Zaid R . The role of zinc finger linkers in zinc finger protein binding to DNA. J Comput Aided Mol Des. 2021; 35(9):973-986. DOI: 10.1007/s10822-021-00413-6. View