Dataset Information

Mass spectrometric ITEM-ONE analysis confirms and refines an assembled affimer binding site on the HER-2 targeting therapeutic antibody Pertuzumab

ABSTRACT: The Pertuzumab-binding affimer 00557_709097 was incubated either with peptides from tryptically digested Pertuzumab or with selected synthesized peptides. Affimer-peptide complexes were subjected to nano electrospray mass spectrometric ITEM analyses in order to identify the affimer’s epitope on Pertuzumab. Furthermore the affimer was incubated with intact Pertuzmab to determine apparent binding energies and dissociation constants of the complex dissociation reactions in the gas phase using ITEM-TWO.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michael Kreutzer

LAB HEAD: Prof. Dr. Michael O. Glocker

PROVIDER: PXD043203 | Pride | 2024-04-09

REPOSITORIES: Pride

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Publications

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

Röwer Claudia C Olaleye Oladapo O OO Bischoff Rainer R Glocker Michael O MO

Biomolecules 20231224 1

Intact Transition Epitope Mapping-One-step Non-covalent force Exploitation (ITEM-ONE) analysis reveals an assembled epitope on the surface of Pertuzumab, which is recognized by the anti-Pertuzumab affimer 00557_709097. It encompasses amino acid residues NSGGSIYNQRFKGR, which are part of CDR2, as well as residues FTLSVDR, which are located on the variable region of Pertuzumab's heavy chain and together form a surface area of 1381.46 Å<sup>2</sup>. Despite not being part of Pertuzumab's CDR2, the ...[more]

PMID: 38254624

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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.

Dataset Information

Mass spectrometric ITEM-ONE analysis confirms and refines an assembled affimer binding site on the HER-2 targeting therapeutic antibody Pertuzumab

Publications

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

Similar Datasets

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