Identification of epitopes by proteolytic cleavage and mass

Mass spectrometry has emerged as a widespread technique for the study of protein structure, function, quantity and interaction with other biomolecules. Important features of the mass spectrometric protein analysis are the high sensitivity, high mass accuracy, short analysis time and low sample consumption. To identify molecules within complex protein mixtures and to dissect the structure of the molecular recognition domains diverse applications have been developed in

conjunction with mass spectrometry. These methods include chromatographic and electrophoretic separations, proteolytic assays, and differential chemical modification of specific amino acid functions, sample preparation and bioinformatic tools for data analysis.

One of the most important applications of MS is that it provides structural identification of epitopes, unlike any of the other methods. The first attempts concerning the investigation of the antigenic determinant resulted by limited proteolytic cleavage of immune complexes were carried out by PAGE [31] and HPLC [32]. However both methods are unsuitable for unambiguous epitope identification. A general, molecular approach for identification of epitopes from peptide and protein antigens using mass spectrometry was developed by our laboratory [33, 34]. This method combines the advantage of the proteolytic stability of antibodies, and the shielding of the epitope with the unambiguous molecular identification provided by MS [16, 35, 36].

For epitope excision, the antigen is digested by proteolytic enzymes following the formation of the immune complex (see Figure 5). The epitope will be resistant to the fragmentation while the peptide sequences exposed to the enzymes will be cleaved.

Cleavage sites of the protease located inside the antigenic determinant that are not fragmented provide information on the epitope. The peptide fragments resulting after cleavage by the proteolytic enzyme as well as the epitope fragments collected after acidic dissociation of the complex are analyzed by mass spectrometry which is capable to provide unambiguous identification of the peptide sequences. In epitope extraction, the proteolytic fragmentation of the antigen in solution yields peptide sequences that might contain the intact epitope if the enzyme has no specificity for the amino acids contained by the antigenic determinant. The resulting peptide fragments are allowed to react with the antibody. Due to the high specificity of the antigen-antibody interaction only the peptides that contain the antigenic determinant in a similar conformation as in the intact protein will interact. The characterization of discontinuous epitopes is usually more difficult involving a combination of proteolytic epitope excision, chemical modification and mass spectrometry [37-41].

a) b)

The methodology for epitope excision using proteolytic cleavage of the unbound peptide sequences followed by mass spectrometric identification of the remaining affinity bound peptides can be employed for the identification of the binding partners of any isolated protein, and therefore, emerged as an important tool in the characterization of protein function [42]. The identification of the specific antigen from a complex mixture of proteins is achieved based on the data base search using the peptide masses determined from the elution fraction. An immobilized anti-troponin antibody and bovine heart cell lysate were used as model system.

A new, recent development, as an analogous method to the determination of epitopes has been the identification of antibody paratope structures using antigen columns containing the immobilized epitope. The antibody is exposed to the antigen column either after digestion in solution (proteolytic paratope extraction), or as an intact molecule that will be digested by a protease after the immune complex is formed (proteolytic paratope excision). The characterization of antibody-paratopes is

Complex peptides are washed off and the affinity bound peptides are dissociated from the antibody.

Both fractions are analysed by mass spectrometry. (b) In epitope extraction, the antigen is first digested in solution, and the proteolytic fragments are allowed to bind to the antibody.

considerably more difficult due to i) higher stability to proteolytic digestion of the antibody compared to the antigen, which makes difficult the application of the proteolytic excision of the paratope; ii) the fact that the antigen binding sites might be scattered within the 6 complementarity determining regions (CDRs) of the heavy and light chains and the individual sequences resulted by digestion in solution during epitope extraction might not display affinity to the antigen and iii) the lack of genomic data for most of the antibodies to provide fast identification of the paratope sequences by comparing the set of peptide masses obtained in the mass spectrum of the paratope elution with the theoretical masses from the sequence database. A first example of paratope identification by mass spectrometry was described for the camel anti-lysozyme antibody cAbLys3 (see Figure 6) [43]. Camel antibodies lack the light chains, therefore containing only three CDRs [44]. The CDR3 is significantly larger than those in human and mouse immunoglobulin and forms an exposed loop of 24 amino acids that fits into the active site cleft of lysozyme [45]. Mass spectrometric paratope identification was applied in this case for a 26 amino acid synthetic peptide containing the CDR3 sequence.

Although mass spectrometry has been an established technique in organic chemistry, the involatility of the macromolecules has limited the applications in the biological and medical field in the past. This difficulty has been overcome by the introduction of “soft”

ionization techniques for effectively dispersing proteins and other molecules into the gas phase with no or little fragmentation. The predominant methods are today i) matrix-assisted laser desorption and ionization (MALDI), and ii) electrospray ionization (ESI).

Lysozyme

cAbLys3

CDR3 Lysozyme

cAbLys3

CDR3

Figure 6: Ribbon representation of the non-covalent complex formed by cAbLys3 camel antibody and hen eggwhite lysozyme (HEL). The CDR3 region of the cAbLys3 is marked in yellow. The structure was prepared with BAllView 1.1 based on the crystal structure with the PDB accession number 1MEL.

In MALDI mass spectrometry applications, the analyte is co-crystallized with a large excess of a matrix. The matrix molecules are typically organic acids which have an absorption in the wavelength at which the laser is used (UV, visible or infrared) [46, 47]. Pulses of laser light (1-10 ns) are applied to the surface of the sample, causing desorption and ionization of the analyte-matrix mixture. The thermal-spike model proposes that the energy absorbed by the matrix molecules causes rapid heating of the irradiated layers of sample followed by evaporation of matrix together with the analyte molecules. The ionization of the analyte occurs probably subsequent to the ejection of the molecules from the support [48]. A schematic representation of the MALDI process is illustrated in Figure 7a. Typically, the mass spectrum of a sample ionized by MALDI contains singly charged molecular ions and ions of low charge states.

In contrast to MALDI-MS, electrospray ionization (ESI) sources operate at atmospheric pressure and provide the transfer of the ions present in liquid samples in the gas phase as isolated entities [49]. Analyte ions are generated by solubilisation in suitable solvents such as acidic aqueous solutions containing methanol or acetonitrile. The analyte solution is pushed through a very small metal capillary. The high electric field applied between the needle and the counter electrode forces the solution to emerge from the tip of the needle giving rise to the so called Taylor cone.

If the electric field is high enough small charged droplets form. The formation of fine droplets from the solution emerging from the needle is facilitated by a sheet flow of nitrogen gas. According to the “ion-evaporation” model, the solvent from each droplet evaporates yielding a higher charge density. When the Coulomb repulsion becomes of the same order as the surface tension, the droplet undergoes fissions producing smaller droplets that also evaporate. The process leads eventually to the formation of droplets containing a single ion. Ultimately fully desolvated ions result from complete evaporation of the solvent.

a)

b)

Figure 7: (a) Schematic representation of the ion formation in MALDI mass spectrometry; (b) Schematic representation of the electrospray process.

The advances in the development of ionization techniques led to an increasing interest in the technological improvements of time-of-flight, ion trap and Fourier transform mass spectrometers for applications in peptide and protein analysis. While FTMS combines all the high performance characteristics (accuracy, resolution, sensitivity), the high cost of magnets and maintenance and the complexity of operation have limited their widespread use to industrial laboratories; however several mass spectrometry research laboratories recently focused on extending the applications and performance of the FTICR-MS methods. Triple quadrupole, TOF and quadrupole ion trap mass spectrometers are three other types of mass analysers with widespread use primarily owing to their cost and ease to use.

nebulising gas

1.4. Pathophysiological characteristics and therapeutic perspectives of