Epitope and Paratope Mapping of a SUMO-Remnant Antibody Using Cross-Linking Mass Spectrometry and Molecular Docking.

Abstract

The small ubiquitin-like modifier (SUMO) is an important post-translational modification that regulates the function of various proteins essential for DNA damage repair, genome integrity, and cell homeostasis. To identify protein SUMOylation effectively, an enrichment step is necessary, often requiring exogenous gene expression in cells and immunoaffinity purification of SUMO-remnant peptides following tryptic digestion. Previously, an antibody was developed to enrich tryptic peptides containing the remnant NQTGG on the receptor lysine, although the specifics of the structural interaction motif remained unclear. This study integrates de novo sequencing, intact mass spectrometry, cross-linking mass spectrometry, and molecular docking to elucidate the structural interaction motifs of a SUMO-remnant antibody. Additional cross-linking experiments were performed using SUMOylated peptides and high-field asymmetric waveform ion mobility spectrometry (FAIMS) to enhance the sensitivity and confirm interactions at the paratope interface. This study establishes a robust framework for characterizing antibody-antigen interactions, offering valuable insights into the structural basis of SUMO-remnant peptide recognition.