A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass Spectrometry Using Secondary Antibodies and Cross-Linking

Peter, Jochen; Tomer, Kenneth B.

doi:10.1021/ac010258n

Cited by 37 publications

(24 citation statements)

References 31 publications

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“…Epitope Mapping with Monoclonal Antibodies-The epitope mapping protocols are based on the approach described by Peter and Tomer (27), which we adapted in two different protocols used here (28).…”

Section: Methodsmentioning

confidence: 99%

Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

Koehler

Carlier

Veggi

et al. 2011

Journal of Biological Chemistry

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NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.

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Section: Methodsmentioning

confidence: 99%

Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

Koehler

Carlier

Veggi

et al. 2011

Journal of Biological Chemistry

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“…CNBr-activated Sepharose beads were activated in accordance with the procedures described previously (45). Briefly, a 20-l aliquot of washed beads was put into each of two CRCs.…”

Section: Antibodies and Reagents (I) Mabsmentioning

confidence: 99%

“…The human 2F5 antibody was affinity captured from solution and cross-linked to the Fc-specific antibody with BS 3 as previously described (45). A solution of 10 mM BS 3 was prepared in pH 7.2 PBS.…”

Section: Antibodies and Reagents (I) Mabsmentioning

confidence: 99%

Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

Parker

Deterding

Hager-Braun

et al. 2001

J Virol

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150

113

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Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

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“…CNBr activated sepharose beads were activated in accordance with the procedures described previously [33,34]. Briefly, a 60 μl aliquot of washed beads was added to each of two CRCs (USB Corporation, Cleveland, OH).…”

Section: Preparation Of Immobilized Antibodiesmentioning

confidence: 99%

“…The beads were incubated for 2 h at room temperature with slow rotation, drained, and washed three times with 0.5 ml of PBS. The CBH-5 HMAb antibody was affinity captured from solution and cross-linked to the Fc-specific antibody with BS3 as previously described [33]. A solution of 10 mM BS3 was prepared in PBS buffer (pH 7.2).…”

Section: Preparation Of Immobilized Antibodiesmentioning

confidence: 99%

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

Iacob

Keck

Olson

et al. 2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Human monoclonal antibodies derived from B cells of HCV infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acids residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.

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A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass Spectrometry Using Secondary Antibodies and Cross-Linking

Cited by 37 publications

References 31 publications

Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

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