3 Experimental Part
3.3 Mass spectrometric methods
A parallel may be drawn between carbohydrate-protein binding and antigen-antibody interactions, considering the carbohydrate analogous to the epitope and the carbohydrate binding site analogous to the paratope. This inspired the major goal of the present dissertation: the development and application of methods similar to paratope excision and extraction mass spectrometry for the general identification of the carbohydrate binding sites (CBS) in proteins. Interactions between proteins and carbohydrates are widely exploited in a variety of chromatographic procedures, such as the affinity capture of lectins on immobilized carbohydrates or isolation and purification of glycoproteins on columns with immobilized lectins. The experimental conditions from such procedures regarding ligand immobilization, protein binding and elution protocols were used as starting points for establishing the new proteolytic- and mass spectrometric-based method for elucidating carbohydrate binding sites in proteins, which are described in this thesis.
The combination of specific proteolytic excision of affinity-bound proteins with mass spectrometric analysis of the eluted peptides has been previously shown to be a highly efficient tool for the identification of interacting protein structures (e.g.
epitope and paratope). There are, however, several differences between protein-protein and protein-carbohydrate interactions in view of binding strength to immobilized ligands and proteolysis. The biggest concern initially came from the low affinity of proteins for carbohydrates. While the interaction strength of a lectin with a mono- or disaccharide is typically in the low millimolar range, it is reasonable to expect that a proteolytic excision approach would yield peptides with an even lower affinity to the carbohydrate than the parent molecule. There are however known cases of both naturally occurring and engineered small peptides able to bind carbohydrates.
Defensins are small (2-6 kDa) cysteine-rich peptides found in plants and animals (both invertebrates and vertebrates), which are involved in antifungal, antibacterial and antiviral host defense [121-123]. Defensins can form oligomers and bind to complex glycans on glycoproteins with high affinities comparable with those of lectins [128].
Also, small proteoglycan-binding peptides (2-3.5 kDa), designed from consensus sequences of heparin-binding proteins, were found to have heparin-affinities in the nanomolar range, which increased with their molecular weight [129].
A first approach developed and employed in the current work for identifying carbohydrate binding sites (CBS) from lectins and other carbohydrate-recognizing proteins was termed CBS-excision [130, 131]. In this method, the carbohydrate is immobilized on an affinity-matrix and incubated with the carbohydrate binding protein (lectin, antibody, etc). The resulting complex is then subjected to proteolytic digestion.
Here, an important difference to the protein-protein excision is due to the proteolytic stability of carbohydrates. Therefore, digestion times may be widely adjusted without adverse effects on the immobilized carbohydrate. In a variation of this approach [130], termed CBS-extraction, the protein is first subjected to proteolytic digestion and the mixture of resulting peptide fragments is subsequently presented to the immobilized carbohydrate. After washing away the non-binding peptides, the remaining affinity-bound fragments are eluted and analyzed by mass spectrometry. A wide range of eluents may be used in both approaches (organic solvents, carbohydrates) and the ligands may be small or complex carbohydrates, as well as glycoproteins and glycolipids. Proteolytic digestion combined with mass spectrometry is a versatile
method, highly suitable for the identification and differentiation of oligosaccharide recognition structures in carbohydrate binding proteins such as lectins.
1.4 Scientific goals of the dissertation
Glycosylation is one of the most common post-translational modifications (PTM), with more than half of the eukaryotic proteins being subject to glycosylation [1]. The recognition of carbohydrates by proteins is one of the most important types of biological interactions, involved in cell differentiation, development, fertilization, pathogen infection, cell-cell recognition, signal transduction, inflammation processes, and cancer cell metastasis. The characterization of these interactions is a crucial step in understanding the mechanisms by which lectins, anti-carbohydrate antibodies and enzymes exert their functions, for defining diagnostic targets and biomarkers, and for the development of therapeutic agents. Although X-ray and NMR have been shown to be highly informative on revealing structural details of carbohydrate recognition structure and binding, their application is limited by the large amounts of high purity material needed, difficulties in obtaining a sufficient resolution, elaborate protocols.
This dissertation is focused on the development of new mass spectrometry-based methods characterized by high specificity, sensitivity and low requirements in sample purity, for the quick identification of carbohydrate recognition structures. The main goals of the dissertation are summarized as follows:
1. Development of new affinity-mass spectrometry approaches for the identification of carbohydrate binding sites in proteins. Using galectin-1 and -3 as test systems, the experimental methods for proteolytic excision and extraction in combination with mass spectrometry were developed. Optimization procedures were carried out regarding proteolysis conditions, buffers and elution conditions.
2. Identification and differentiation of the carbohydrate binding sites in several galectins. The analysis of the primary structure of human galectins (galectin-1, -3, -4 and -8), chicken galectin-3 and rat galectin-5 was performed by proteolytic peptide
mapping - mass spectrometry. The carbohydrate binding sites in galectins were elucidated using the newly developed affinity-mass spectrometry methods. A further goal was the comparison of binding specificities and differentiation of blood group oligosaccharide recognition between human and chicken galectin-3.
3. Evaluation of the carbohydrate recognition properties of galectin-derived synthetic peptides. The amino acid sequences of galectin peptides involved in carbohydrate binding were used as starting point for the design of corresponding peptides. The interactions of the synthetic peptides with carbohydrates were characterized both by affinity-mass spectrometry and by SAW biosensor analysis.
4. Elucidation of the carbohydrate binding site in human alpha-galactosidase A. The newly established affinity-mass spectrometry methods were employed for the identification of galactose binding site in alpha-galactosidase A, a key lysosomal enzyme for Fabry's disease. A main goal was to study the influence of several pharmacological chaperones on galactose recognition by alpha-galactosidase A.
2 Results and Discussion
2.1 Proteolytic-excision and -extraction mass spectrometry for identification of carbohydrate recognition sites in proteins
The principle of proteolytic-excision and combined with mass spectrometry for the identification of carbohydrate recognition sites in proteins is presented in Figure 10. First, an affinity column is prepared by immobilizing a carbohydrate on a solid support. The carbohydrate may be a mono-, di-, or oligosaccharide (branched or linear) immobilized directly on the support material via a hydroxyl group.
Alternatively, a glycoconjugate can be used and the coupling performed through a functional group of the aglycone. The carbohydrate binding protein (lectin, antibody, etc.) or mixture of proteins is added over the affinity column and is incubated, to allow the formation of the protein-carbohydrate complex. The conformation of the protein can be kept in native state by performing the experiment under physiological conditions. A washing step using the binding buffer usually follows, to remove excess unbound protein, non-binding proteins and other impurities. Only the affinity-bound protein remains on the column and it is subsequently subjected to proteolytic digestion. The protease is chosen considering the sequence of the target protein and the length of the resulting theoretical peptides. By using different proteases, overlapping peptides may be obtained and varying digestion times may lead to the generation of partial peptides. The proteolysis step may be prolonged for as long as required, since carbohydrates are inherently stable to the action of proteases. These strategies enable a precise identification of carbohydrate binding sites. In the following step, the resulting protein fragments not binding to the carbohydrate are washed away from the column. The remaining affinity-bound fragments are eluted using a carbohydrate solution (competitive elution) or a mixture of volatile organic solvents.
All recovered fractions are then analyzed by mass spectrometry.
In the extraction approach, the protein is first digested in solution and the resulting peptide mixture is subsequently added over the immobilized carbohydrate.
Peptides presenting affinity towards the carbohydrate will bind to it. After washing away the non-binding fragments the affinity-retained peptides are eluted and analyzed by mass spectrometry. In both approaches, the washing steps before elution ensure that only carbohydrate-specific peptides remain on the column. The last washing fraction is analyzed by mass spectrometry to confirm the complete removal of non-binding peptides and further washing steps may be employed as needed. After elution, the affinity matrix is regenerated by washing with binding buffer to prepare the column for further use.
Figure 10. Analytical approaches for mass spectrometric identification of carbohydrate binding structures in proteins. (a) In the excision approach the carbohydrate binding protein (lectin, antibody, etc) is bound on a column with immobilized carbohydrate and digested with proteases. The protein fragments not binding to the carbohydrate are washed from the column and the remaining bound fragments are eluted. (b) In the extraction approach the protein is first digested in solution and the resulting fragments are bound to the carbohydrate. The non-binding peptides are removed by washing and the binding fragments are recovered.
A wide range of solid supports (matrices), usually available as microspheres, can be employed for carbohydrate immobilization and are commercially available either in unmodified form, activated form or with pre-immobilized carbohydrates. The matrices may include agarose derivatives (Sepharose, Seralose) [132-138], dextran (Sephadex) [139-141], silica carriers (Synsorb) [142, 143], iron oxide (magnetic beads) [144, 145] and a large variety of polymers like polyacrylamide [146, 147], poly-2-hydroxy-ethyl methacrylate (Spheron) [148, 149], ethylene glycol/methacrylate copolymers (Tyopearl) [150]. Carbohydrates can also be immobilized on microtiter plates [151, 152], membranes and various biosensor and microarray supports. The number and type of available immobilization supports and chemistries increase if the carbohydrate is immobilized as a glycoside. If the carbohydrate is immobilized as a glycoconjugate and the excision approach is applied, then the carrier's proteolytic susceptibility has to be taken into account and evaluated beforehand.
The most widely used matrix is an agarose-based bead-formed material known under the trademark of Sepharose. In the present work Sepharose 4B and Sepharose 6B containing 4 % and 6 % (w/v) agarose, respectively were employed. Agarose is a linear polymer of agarobiose (Figure 11), which is a disaccharide composed of D-galactose and 3,6-anhydro-L-D-galactose (systematic name: β-D-galactopyranosyl-(1→4)-3,6-anhydro-α-L-galactopyranose). Sepharose 6B is commercially available as epoxy-activated Sepharose, while Sepharose 4B was activated with divinyl sulfone (Figure 11).
sulfone used for matrix activation. (c) Structure of lactosylated Sepharose with divinyl sulfone activation.
For immobilization of small carbohydrates it is usually necessary to introduce a spacer between the ligand and the matrix, to allow accessibility to the lectin's binding site. The spacer may be linked to the matrix before immobilizing the carbohydrate or the carbohydrate may be converted into a glycoside by derivatizing it with the spacer. The matrix activation step may be combined with the linker insertion.
Heterobifunctional linkers may be used to change the available matrix chemistry and introduce active groups favorable to carbohydrate immobilization. After the carbohydrate immobilization, blocking of any remaining active groups on the matrix should be done, usually with small organic molecules (e.g. ethanolamine). Details on the blocking procedure employed for the matrices used in this work are given in the experimental section.
Since matrices based on polysaccharides (e.g. Sepharose) can bind lectins, affinity experiments on such matrices may lead to the isolation of proteins and peptides specific for the matrix in addition to those specific for the immobilized ligand. In order to eliminate false-positive responses it is necessary to carry out rigorous control experiments. These controls may be performed using the
underivatized matrix (with blocked active groups) or with an immobilized carbohydrate for which the target proteins or peptides have no affinity and to which they would theoretically not bind.
2.1.1 Development of elution systems for dissociating carbohydrate protein/peptide complexes
Affinity capture of lectins and other carbohydrate-binding proteins on immobilized carbohydrates is routinely employed for protein isolation and purification. After incubating a protein mixture with a carbohydrate immobilized in an affinity column, the protein that recognizes the carbohydrate binds to it with an yield depending on its affinity and the experimental conditions. The carbohydrate column is then washed free of unwanted proteins and the protein bound to the carbohydrate is eluted. A second application exploiting the specificity of lectin-carbohydrate interactions is Lectin Affinity Chromatography, which is used for the isolation and purification of glycoproteins from protein mixtures, on columns with immobilized lectins. Glycoproteins with sugar chains recognized by the immobilized lectin bind, non-binding (glyco)proteins are removed by washing and the retained glycoproteins are eluted.
For both described methods the typical analytical workflow is identical and elution of the target protein is usually performed with a solution of a lectin-specific free sugar. Lectins bind carbohydrates noncovalently, reversibly and relatively weakly, at least in the case of mono- and disaccharides, which are the most widely used ligands. Therefore, (glyco)proteins may be readily released from the affinity column due to the competition between the free and immobilized sugars. Solutions are usually kept at about neutral pH, to maximize the yield of protein recovery and to preserve the activity of the column for repeated use. In some cases achieving complete elution with a sugar solution may be difficult, especially for the elution of glycoproteins from immobilized lectins, due to the complexity of the glycans that usually translates into a stronger bond to the lectin. Depending on the lectin-carbohydrate system employed and its affinity constant, the elution may require more complex and expensive sugars.
Eluted proteins are detected by measuring the absorbance at 280 nm and by following the agglutinating activity of the flow-through. The recovered (glyco)protein solution contains a significant amount of carbohydrate that must be removed, usually through dialysis. If the immobilized lectin (or the lectin to be purified) is dependent on cations (usually Mg2+, Ca2+, Mn2+) for its activity, elution may be achieved by removing the cations from the buffers passed through the affinity column. Most carbohydrates and lectins are stable and can tolerate a wide range of pH and ionic strengths. Therefore, elution may be performed by carefully choosing conditions that disrupt intermolecular protein-carbohydrate bonds, but do not irreversibly damage the proteins or hydrolyze the sugar. The problems associated with separation systems based on protein-carbohydrate interactions may be avoided by recombinant expression of the target protein, with inclusion of an amino acid sequence tag followed by purification through more manageable affinity procedures.
In the case of excision and extraction methods for the identification of carbohydrate binding peptides, several elution systems were developed that completely remove the bound proteins or peptides, do not damage the affinity matrix and allow the column to be reused. These elution systems were tested in various experimental setups, using galectins and synthetic galectin-derived peptides. The results were evaluated by one dimensional gel electrophoresis (1D-SDS-PAGE) for the proteins (see Chapter 2.3), mass spectrometry and surface acoustic waves (SAW) biosensor for the peptides.
Competitive elution was performed using a concentrated solution of the same carbohydrate immobilized on the affinity column and was therefore a specific elution.
For the experiments with immobilized lactose, affinity-bound galectins or galectin peptides were eluted under strong shaking with 400 μL 0.3 M lactose in 50 mM phosphate buffer with 50 mM NaCl, pH 7.5 at 37 °C for 15 min. The procedure was repeated once and the two elution fractions were mixed and lyophilized. Due to the high concentration of carbohydrate employed, sample desalting prior to MS analysis was carried out even for LC-MS. When required, DTT (1 g/L) was added to the eluent and binding buffer to prevent the formation of intermolecular peptide dimers through
disulfide bonds formation. This had no adverse effects on the affinity of galectins and galectin peptides.
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Figure 12. Affinity-MS of synthetic hGal-1[37-48] on immobilized lactose, using lactose elution. In addition to the protonated peptide ([M+H+]+) in the mass spectra of the supernatant (a) and of the elution (c) the protonated disulfide dimer ([D+H+]+) may be observed. The formation of the dimer may be attributed to the acidic solvent employed for sample desalting and for the MALDI matrix. The MS of the washing fraction (b) was clean, as demonstrated by the lack of peptide signals.
Figure 12 shows the results of an affinity-MS experiment of synthetic galectin peptide hGal-1[37-48] on immobilized lactose, for which 0.3 M lactose solution was used as eluent. This peptide was identified through excision and extraction experiments as part of the lactose binding site in human galectin-1 (see Chapter 2.4.1).
Its synthetic equivalent was shown by affinity-MS and SAW biosensor analysis to bind to lactose and was therefore chosen to study the efficiency of the tested elution
systems. All fractions were desalted prior to MS measurements using reversed-phase pipette tips (see Chapter 3.6.2). The mass spectrum of the washing fraction was clean, indicating that the peptide recovered in the elution fraction was specifically bound. In addition to the protonated peptide ([M+H+]+) in the mass spectra of the supernatant and the elution fractions the protonated disulfide dimer ([D+H+]+) may be observed.
The dimer formation was most likely due to the acidic solvents employed for elution, sample desalting and MALDI matrix preparation (see Chapter 3.3.1).
Several affinity experiments were carried out on immobilized A and B blood group tri- and tetra-saccharides. In these cases the elution with lactose was unsuccessful, even with 0.6 M (close to saturated) solutions. This may be explained on one hand by the inability of lactose to compete with the A and B oligosaccharides for binding of peptides and on the other hand by lactose interference with the desalting procedure. The high cost of complex sugars discouraged employing competitive elution with the same sugars. Therefore, several elution systems employing organic co-solvents and compounds were developed and tested. The most successful systems were based on acetonitrile, such as 60 % ACN, 0.1 % TFA in water, pH 2 (Figure 13) or 80 % ACN in water (pH 7). The elutions were performed in a similar manner to the competitive elution, by adding 400 μL eluent, shaking for 15 min. and repeating the procedure once. To test the effectiveness of the elution systems employing organic solvents, a second elution was carried out using 0.3 M lactose. Competitive elution is an established procedure and can be used as a standard to assess the effectiveness of other elution systems. The lack of MS signals from the lactose elution indicated that the first elution was successful (Figure 13).
Since a dehydrating effect of acetonitrile-based eluents on the Sepharose affinity matrix was observed, equilibration with binding buffer was performed immediately after the elution step. The dehydrating effect was proportional to the concentration of ACN employed, but the immediate re-equilibration enabled the matrix to recover and the columns to be reused successfully. The use of 100 % ACN (Figure 15) completely dehydrated and irreversibly damaged the affinity matrix. Other elution systems such as 100 mM acetic acid (Figure 14) or 5-10 % ACN in water did
not perform equally well, while 0.1 % aqueous TFA or solutions based on non-volatile substances such as glycin or chaotropic agents were not successful or required subsequent desalting.
The properties of the elution system were carefully considered so that sensitive glycosidic linkages would not be hydrolyzed. For example, the glycosidic bond of sialic acids (O- and N substituted neuraminic acid) is sensitive to acidic hydrolysis [153]. O-acetyl groups in neuraminic acid derivatives are sensitive to basic conditions which can lead to de-acetylation and O-acetyl migration [154]. In order to prevent the deglycosylation or changes in carbohydrate structure, when working with sialylated
The properties of the elution system were carefully considered so that sensitive glycosidic linkages would not be hydrolyzed. For example, the glycosidic bond of sialic acids (O- and N substituted neuraminic acid) is sensitive to acidic hydrolysis [153]. O-acetyl groups in neuraminic acid derivatives are sensitive to basic conditions which can lead to de-acetylation and O-acetyl migration [154]. In order to prevent the deglycosylation or changes in carbohydrate structure, when working with sialylated