MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies

Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.     

Actinomycin D inhibition of monoclonal antibody binding to nucleolar phosphoprotein 37/5.2 (B23)

Actinomycin D was found to block binding of a monoclonal antibody to nucleolar phosphoprotein 37/5.2 (B23) when incubated either simultaneously with or prior to addition of the antibody. Daunorubicin had no significant blocking activity of this antigen-antibody reaction. In comparative studies with a monoclonal antibody to nucleolar phosphoprotein 110/5.2 (C23), actinomycin D exhibited little blocking activity. When a 42-mer peptide containing the epitope of protein 37/5.2 (B23) was tested as the antigen, similar inhibition by actinomycin D of binding of the monoclonal antibody was found. These results provide evidence for binding of actinomycin D to a specific epitope of protein 37/5.2 (B23).     

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.     

Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.     

Identification of cross‐reactive B‐cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI‐TOF MS

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross‐allergenicity described between soy and milk proteins. We have previously identified several cross‐reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1‐casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α‐casein‐specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross‐reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI‐TOF MS analysis. On a second approach, the peptide mixture was resolved by RP‐HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI‐TOF MS. This novel MS based approach led us to identify and characterize four peptides on α‐casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross‐reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross‐reactivity, to further develop new and more effective vaccines for food allergy.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes.

An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones.     

Enzymatic splitting of antibodies bound to immobilized antigen as a means of Fab fragments preparation]

A method of Fab fragments preparation by enzymatic splitting of antibodies bound to specific antigen immobilized on an insoluble support is described. The complex of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD), immobilized on Sepharose 4B, with anti-rat GAPD rabbit antibodies was digested with papain. The antigen was inaccessible to proteolysis under conditions employed. After 4 hrs of incubation with papain the antibody was completely split into non-precipitating fragments. The products of proteolysis not bound to Sepharose, were eluted with 0.1 M givcine buffer pH 2.5, and shown to correspond to Fab fragments.     

A monoclonal antibody to cyclomaltoheptaose (β-cyclodextrin): Characterization and use for immunoassay of β-cyclodextrin and its derivatives

Cyclomaltoheptaose (β-cyclodextrin, β-CD) is a promising compound for application in various industrial fields because of its ability to entrap various compounds into its hydrophobic cavity. A monoclonal antibody (A7) to β-CD was generated by using a conjugate of glucosaminylmaltosyl-β-CD and bovine serum albumin as an antigen. The A7 monoclonal antibody was IgM/κ and reacted with β-CD with high specificity. The epitope recognized by the A7 monoclonal antibody seemed to be located on the secondary hydroxyl groups of the rim side of the β-CD molecule. The dissociation constant of the complex of β-CD and the immobilized A7 monoclonal antibody was determined to be 1.2 × 10^-4 M. A competitive ELISA using the A7 monoclonal antibody enabled determination of β-CD and its derivatives with a detection limit of 0.05 μM. This immunoassay was useful to determine β-CD in biological fluids such as human plasma and urine after appropriate pretreatment of the samples. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid and sensitive identification of major histocompatibility complex class I-associated tumor peptides by Nano-LC MALDI MS/MS

Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.     

Characterization of epitopes on the cationic peanut peroxidase by four monoclonal antibodies

The epitope sites on the cationic peanut peroxidase were characterized by four monoclonal antibodies raised against this isozyme. Evidence is presented showing that the epitope for monoclonal antibody 1B is located on the polypeptide. Sensitivity of the epitopes recognized by 1M and 2F to 0.1M HCl, boiling, 10 mM periodate and trifluoromethane sulfonic acid treatment indicate that they occur at regions where oligosaccharides are linked to the polypeptide backbone. The antigenic specificity of 2A is, in addition, dependent on the conformation of the epitope site which is destroyed after partial proteolysis of the peroxidase.     

Recognition of Mesothelin by the Therapeutic Antibody MORAb-009: STRUCTURAL AND MECHANISTIC INSIGHTS*

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.     

Identification of the epitope for anti-cystatin C antibody

Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.     

Long-range changes in a protein antigen due to antigen-antibody interaction.

Amide exchange kinetics were used to probe the conformation of hen egg-white lysozyme complexed with the anti-lysozyme monoclonal antibody HyHEL-5. Following the technique developed by Paterson et al. [(1990) Science 249, 755-759] we used two-dimensional NMR to measure amide exchange kinetics of the lysozyme amide protons in the lysozyme-antibody complex. A total of 15 amide protons showed altered exchange kinetics in the presence of the complex. Five of these 15 protons reside on residues that are found within the epitope as defined by X-ray crystallography. Five residues are located at the perimeter of the epitope. The remaining five residues are removed from the epitope. The perturbation of amide exchange rates at sites distant from the epitope indicates that the formation of antigen-antibody complexes can produce changes in the antigen at sites that are quite distant from the structural epitope.     

Mapping of four discontiguous antigenic determinants on horse cytochrome c

The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibody-bound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double-labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen-antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5-fold to over 20-fold lower reactivity in antibody-bound cytochrome c. The present results are complemented by previous data obtained by cross-reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformation-dependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information.     

Molecularly imprinted polymers coupled to matrix assisted laser desorption ionization mass spectrometry for femtomoles detection of cardiac troponin I peptides

Molecularly imprinted polymers (MIPs) were combined to MALDI‐TOF‐MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7–2μm), dissociation constants (58–817 nM), kinetics of binding (5–60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI‐TOF‐MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP‐SE MALDI‐TOF‐MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP‐SE MALDI‐TOF‐MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes. Copyright © 2015 John Wiley & Sons, Ltd.     

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.