Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Abstract

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and highresolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures

“…They lead to efficient dissociation of the antibody-antigen complex presumably by destroying the tertiary structures of the proteins, that is, the antigen and the antibody (Fornsted, 1984). This elution principle has been applied for mass spectrometric epitope mapping (Suckau et al, 1990;Macht et al, 1996;Hager-Braun et al, 2006;Stefanescu et al, 2011) and related applications (Stefanescu et al, 2007;Popescu et al, 2008;Jimenez-Castells et al, 2012). Nevertheless, depending on the complexity of the antigen-containing solution, for example, patient serum, the eluted target molecules (antigens) are usually accompanied by unspecifically bound "background" molecules that arise from nonspecific interactions of (unknown) molecules with the bead surfaces and those of the capturing molecules, respectively.…”

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

“…For the identification of peptide and protein interaction structures, affinity-MS in combination with selective proteolytic digestion (epitope excision) and affinity selection of proteolytic fragments (epitope extraction) has been developed and effectively applied in a number of previous studies [16][17][18][19][20][21]. Here, we report the identification of the interaction epitopes and binding affinity of the complex between HN and Aß(1-40) using affinity-MS, biosensor analysis and ELISA and perform a structural characterization of the complex using molecular docking simulation.…”

Automatic Determination of Disulfide Bridges in Proteins

“…When the sample is abundant, the disulfide-linked peptides may also be identified by using direct infusion using the syringe pump, bypassing the HPLC run. LC-MS/MS analysis of the disulfide-linked peptides using DDA involves analysis of the peptide mixture under both NR and R. 9,12,19,26,29,[43][44][45][46][47][48] In the first LC-MS/MS run, the peptide mixture contains peptides with their Cys in reduced form and alkylated by an alkylating agent such as iodoacetamide (IAA) or iodoacetic acid. Therefore, the Cys-containing peptides can be identified and used as an indicator of what Cys-containing peptides may be identified in a LC-MS/MS run as both single peptides or as part of a disulfide bridge.…”

“…For example, misfolding of a secreted protein with scrambled disulfide bridges will prevent it from performing its cellular or extracellular (or physiological) function. 3,8,10,[12][13][14][15][16][17][18][19][20][21][22][23][24][25] In addition, a point mutation that would replace a cysteine (Cys) amino acid to a different one, thus preventing disulfide formation, would not only prevent the mutated protein from performing its function but also enable pathological protein-protein interactions and aggregations based on the free, unpaired Cys from the predicted disulfide bridge. 1,3,26,27 Therefore, identification of disulfide bridges allows us to understand not only the structure of proteins but also their physiological and pathological states.…”