Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

Rinaldi, Francesca; Lupu, Loredana; Rusche, Hendrik; Kukačka, Zdeněk; Tengattini, Sara; Bernardini, Roberta; Piubelli, Luciano; Bavaro, Teodora; Maeser, Stefan; Pollegioni, Loredano; Calleri, Enrica; Przybylski, Michael; Temporini, Caterina

doi:10.1007/s00216-018-1466-z

Cited by 15 publications

(15 citation statements)

References 30 publications

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“…ITEM-THREE differs from MALDI MS-based epitope mapping methods (20,(53)(54)(55) in several aspects. Most currently available MALDI-MS approaches for epitope mapping require immobilization of the capturing antibody on a protein A (or protein G) substrate or on some other sort of a substrate (beads or columns) when chemically immobilized.…”

Section: Discussionmentioning

confidence: 99%

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

Danquah

Röwer

Opuni

et al. 2019

Molecular & Cellular Proteomics

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Section: Discussionmentioning

confidence: 99%

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

Danquah

Röwer

Opuni

et al. 2019

Molecular & Cellular Proteomics

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“…To simplify the sample preparation, a protein cocktail purified from ECO157 cell lysates was used for IAC in this study. In many previous epitope identification studies, recombinant protein samples were usually used (47 out of 52 studies), although native protein (3 out of 52 reports) and cell lysates (2 out of 52 reports) were also adopted (Opuni et al 2018 ; Rinaldi et al 2019 ). The detection of some impurities like lpp (8.3 kDa, pI 9.3) and OmpA (37.2 kDa, pI 5.99) with nonmatched pI values and MWs in this study indicated that there was non-specific binding with the relatively complex cell protein samples, which may complicate the analysis of results from affinity mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Wang¹,

Zhou²,

Sang³

et al. 2021

Appl Microbiol Biotechnol

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The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Key points • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC–MS/MS. • The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.

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“…The SPR-MS interface provides sample concentration and in situ desalting for direct MS analysis of the ligand eluate. , Both ESI-ion trap and triple quad-MS systems can be coupled to the SPR-MS interface. Applications of the SPR-MS combination were previously described in studies of a mixed-disulfide antibody epitope of the rheumatoid target protein, HLA-B27, and the epitope identification of chaperone complexes of lysosomal enzymes. ,, …”

Section: Methodsmentioning

confidence: 99%

Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry

Mihoc

Lupu

Wiegand

et al. 2020

J. Am. Soc. Mass Spectrom.

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Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight αhelices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant K D of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146−153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.

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Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

Cited by 15 publications

References 30 publications

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry

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