2 Results and Discussion
2.1 Proteolytic-excision and -extraction mass spectrometry for
2.1.1 Development of elution systems for dissociating carbohydrate
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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[M+H]+
[D+H]+ hGal1[37-48]
DSNNLCLHFNPR
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[D+H]+
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[M+H]+
Supernatant
Wash
Elution a)
b)
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[M+H]+
[D+H]+ hGal1[37-48]
DSNNLCLHFNPR
2856.3 1429.4
[D+H]+
2856.1 1429.5
[M+H]+
Supernatant
Wash
Elution a)
b)
c)
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 carbohydrates, neutral solutions were employed while keeping the elution time to a minimum.
Elution 1
Elution 2 [M+H]+
[D+H]+ 2856.5 1429.3
[M+H]+ 1429.3
[D+H]+ 2856.4
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Supernatant
Wash hGal1[37-48]
DSNNLCLHFNPR
a) c)
b) d)
Elution 1
Elution 2 [M+H]+
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[M+H]+ 1429.3
[D+H]+ 2856.4
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Supernatant
Wash hGal1[37-48]
DSNNLCLHFNPR
a) c)
b) d)
Figure 13. Affinity-MS of synthetic hGal-1[37-48) on immobilized lactose, using ACN/TFA elution.
The mass spectrum of the supernatant (a) shows monomer and dimer signals. The washing fraction (b) was clean. The elution with ACN:0,1 % TFA 2:1 (c) was successful. The second elution (d) using lactose did not show any peptide signals, indicating that the previous elution (c) was complete.
[D+H]+ 2856.5 [M+H]+
1429.4
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[D+H]+
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Elution 1
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Elution 2
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[D+H]+
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Elution 1
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Elution 2 hGal1[37-48]
DSNNLCLHFNPR
a) c)
b) d)
Figure 14. Affinity-MS of synthetic hGal-1[37-48) on immobilized lactose, using 100 mM acetic acid elution. The mass spectrum of the supernatant (a) shows monomer and dimer signals. The washing fraction (b) was clean. The elution with 100 mM acetic acid (c) worked but only partially, as shown by the second elution with lactose (d), which produced strong signals of the synthetic peptide.
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[D+H]+
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[D+H]+
Figure 15. Affinity-MS of synthetic hGal-1[37-48) on immobilized lactose, using 100 % ACN elution.
The mass spectrum of the supernatant (a) shows monomer and dimer signals. The washing fraction (b) was clean. The elution with 100% ACN (c) was successful. The second elution using lactose (d) did not produce any signals, indicating that the previous elution (c) was complete. The elution with 100 % ACN completely dehydrated the Sepharose and subsequent affinity experiments on the same column failed.
In contrast to carbohydrate-based eluents, the eluents based on organic solvents are unspecific and, depending on type and concentration, are able to remove both specifically and unspecifically bound material. Therefore, careful washing steps with binding buffer were used prior to the elution. The number of washing steps was adjusted to ensure that the last washing fraction was clean. To ascertain that only specific peptides were identified, carefully designed control experiments were performed using (i), the underivatized affinity matrix and (ii), an immobilized carbohydrate to which the peptide or protein of interest had no affinity. The use of control experiments was especially important since the matrix employed (Sepharose) is polysaccharide-based, and therefore could bind lectins.
2.2 Structural characterization of galectins by proteolytic peptide