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a Amino acid sequence of bPrP(25-241). b, c Diagrams displaying the mean values with the standard error of the mean of the absolute peak intensities for each of the identified bPrP(25- 241) peptide in the supernatant (gray bars) and in the elution fractions (black bars), shown on a logarithmic scale. The data were obtained from three independent experiments on chymotryptic epitope excision–mass spectrometry on a sepharose column b with immobilized mAb3E7 and c without the antibody immobilized. There were no bPrP peptides detected in the washing fraction before elution. Each peptide was identified at least in two experiments and depicted with its numbers assigned corresponding to the position in the bPrP sequence and with its charge state. Signal intensities below 500 arbitrary units were considered as noise
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The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves...
Citations
... It can monitor specific conformation changes of antibodies and their dynamics in solution and provide epitope−paratope mapping. 14 It utilizes several experimental approaches, such as epitope excision, 15 epitope extraction, 16 chemical footprinting, 17 hydrogen−deuterium exchange (HDX), 18,19 chemical cross-linking, 20 or fast photochemical oxidation of proteins (FPOP). 21 The recent discovery of techniques utilizing the radical fluoroalkyl for protein footprinting 22−25 offers another approach for the structural characterization of therapeutic antibodies and identifying epitope−paratope interacting regions. ...
Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen–antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.
... For obtaining the identification of peptide(s) epitopes two approaches epitope excision [27,28] and epitope extraction [29], both using mass spectrometric analysis of the resulted immunoaffinity chromatographic fractions have been developed. Briefly, in epitope excision approach, identification of the epitope peptide released from an immobilized antibodyantigen complex is done after proteolysis performed on the immunoaffinity column, followed by mass spectrometric measurements [22,30,31]. On the other hand, in the epitope extraction approach the protease-digested antigen peptide mixture is bound to an immobilized antibody and the epitope peptides are eluted and analyzed by mass spectrometry after multiple washing steps [30]. ...
Identifying antigen–antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen–antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen–antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.
... Polyethylene glycol (PEG) precipitation is commonly used to extract ICs from biological fluids. PEG works effectively for precipitating ICs exclusively from other biomolecules [43]. PEG separately precipitates immunoglobulins and ICs, depending on its concentration. ...
... Among them, X-ray crystallography and nuclear magnetic resonance spectrometry precisely determine the structures of ICs at the atomic level. However, these methods are not in routine use because only a small fraction of Ab-Ag complexes can be crystallized and nuclear magnetic resonance has recently limited utility for the study of large proteins [43,68]. Mass spectrometric epitope mapping has become a powerful tool for in vitro epitope mapping because of its high sensitivity and accuracy [67]. ...
Immune complexes (ICs) formed by foreign or self-antigens and antibodies in biological fluids affect various tissues and are thought to cause several diseases. Biological and physical properties of IC, abnormal IC amounts, IC deposition and their relationships with disease pathogenesis had been studied. However, the relationship between ICs and each disease is not well understood and little is known of what determined ICs deposition in particular organ and why different organs are affected in different diseases. Recent technological advance enables identification of ICs in particular its antigens in tissues and body fluids, which may provide a key to discover an important trigger for immunological abnormality occurrence. Further identification of their epitopes, that are the exact origin of antigenicity, is developing and may be useful for diagnosis, elucidation of pathogenesis and treatment against IC-induced diseases. Here, we first make an overview of clearance of ICs, IC-induced pathogenesis and biological properties of ICs. Then, we introduce various methods developed to recover ICs from biological fluids or to identify antigens incorporated into ICs. Furthermore, several methods that can be used in epitope mapping for IC antigens are also documented.
... Immobilization was mostly carried out such that the antibody was covalently attached to a carrier material or a solid support. Carrier materials were tresylactivated sepharose (Suckau et al., 1990), agarose gels (Papac, Hoyes, & Tomer, 1994;Griffiths et al., 2011), polystyrene beads (Pimenova et al., 2009), magnetic microcellulose beads (Yu, Gaskell, & Brookman, 1998), Perloza MT 500 carriers (Jankovicova et al., 2008), sepharose beads (20 papers; therefrom CNBr-activated: 12 papers). The use of so many different carrier materials represents the versatility of epitope mapping procedures and the associated adaptability of the method to particular in-solution handling conditions, if necessary. ...
Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev.
... Epitope excision combined with mass spectrometry is the a good method of epitope identification for a wide variety of proteins (e.g. prions [77], inhibitors [78,79] and lectins [80,81]). ...
... In terms of mass spectrometric epitope-mapping procedures, epitope excision and/or extraction have been the methods of choice for studying such noncovalent molecular antigen antibody interactions. [9][10][11][25][26][27] The antibody/peptide ratio differed from report to report including examples where excess of antibody over the antigen varied over broad ranges, [12,13,25,26,28] whereas in other studies, antigens were used in excess to antibody. [9,29,30] In most of these published methods, fairly large amounts of antibodies (about 300-6000 pmol) were consumed. ...
... In terms of mass spectrometric epitope-mapping procedures, epitope excision and/or extraction have been the methods of choice for studying such noncovalent molecular antigen antibody interactions. [9][10][11][25][26][27] The antibody/peptide ratio differed from report to report including examples where excess of antibody over the antigen varied over broad ranges, [12,13,25,26,28] whereas in other studies, antigens were used in excess to antibody. [9,29,30] In most of these published methods, fairly large amounts of antibodies (about 300-6000 pmol) were consumed. ...
We demonstrate the development of a mass spectrometry-based epitope-mapping procedure in combination with Western blot analysis that works also with antigens that are insoluble in nondenaturing buffers consuming minute amounts of antigen (approximately 200 pmol) and antibody (approximately 15 pmol), respectively. A polyclonal anti-TRIM21 rabbit antibody serum is applied as a model serum for future patient analyses to set up the system. The major epitope that is recognized by the anti-TRIM21 serum spans the central TRIM21 region LQ-ELEKDEREQLRILGE-KE, showing that immunization with a 139-amino acid residue long peptide resulted in a 'monospecific' polyclonal antibody repertoire. Protein structure investigations, secondary structure predictions, and surface area calculations revealed that the best matching partial sequence to fulfill all primary and secondary structure requirements was the four amino acid spanning motif 'L-E-Q-L', which is present in both the sequential and the α-helical peptide conformation. Peptide chip analyses confirmed the mass spectrometric results and showed that the peptide chip platform is an appropriate method for displaying secondary structure-relying epitope conformations. As the same secondary structures are present in vivo, patient antibody screening, e.g., to identify subgroups of patients according to distinct epitope antibody reactivities, is feasible. Copyright © 2013 John Wiley & Sons, Ltd.
... The principle of epitope identification has been developed since the early 1990's in our laboratory [168,311,[315][316][317][318][319] , by combining isolation of antibody-bound peptides using immuneaffinity techniques followed by the precise identification of epitope peptides by mass spectrometry (s. 3.7.2) [254] . ...
... In epitope excision studies, mass spectrometry was used to identify epitope peptides released from an immobilized antibody-antigen complex after limited proteolysis (Pimenova et al., 2009;Parker and Tomer, 2002;Suckau et al., 1990). Here, the antibody prevents either proteolysis (Jemmerson, 1996) or chemical modification (Burnens et al., 1987) of sites of the antigen that are situated in the antibody binding pocket. ...
... Our experimental procedure shows that incubation at a 3:1 ratio of antibody to peptides is well suitable for epitope mapping. This rather fixed antibody/peptide ratio differs from those reported in the literature where excess of antibody varies in broader ranges (Pimenova et al., 2009;Dhungana et al., 2009;Zhao and Chait, 1994) while in other studies peptides were used in excess (Bílková et al., 2005;Parker and Tomer, 2002;Peter and Tomer, 2001;Kiselar and Downard, 1999;Papac et al., 1994). ...
The development and application of a mass spectrometry-based epitope mapping procedure in solution and without immobilization of the antibody is described. Antigens were digested with proteases. Then size exclusion micro-column chromatography (SEC) was carried out prior to and upon exposure of the peptide mixtures to monoclonal antibodies. The epitope-containing peptide, affinity bound to the antibody and, thus, forming a stable complex eluted early as did all high-mass components, whereas all unbound low-mass peptides eluted late. Comparison of elution profiles in the presence and absence of the antibody showed a shift only for epitope-bearing peptides, enabling direct identification of the epitope. His-tag containing recombinant antigens Fibrillarin and RA33 were used in combination with an anti-His-tag monoclonal antibody in order to develop the method. Application of this method for the determination of an epitope on RA33 against which a monoclonal antibody was directed identified the epitope sequence (85IDGRVVEPKRA95) using MS/MS peptide sequencing. Advantages of this approach include low sample consumption, few handling steps, and short duration of analysis. With our method we are ultimately aiming at developing a screening procedure to identify major epitopes in patients that may be suitable in the future for stratification of patients, needed for personalized therapies.
Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP(C)) into a prion (PrP(Sc)). The information necessary for this conversion is contained in the conformation of PrP(Sc). Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10(-18)mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP(Sc) and GPI-anchorless PrP(Sc). It has also been used to study the quaternary structure of the PrP(Sc) multimer. Small molecule reagents react differently with the same lysine in the PrP(C) conformation than in the PrP(Sc) conformation. Such differences can be detected by western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrP(Sc) without the need for proteinase K pretreatment and can be used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions.
