Identification and quantification of tyrosine-nitrated peptides-antibody

exchange mass spectrometry

Protein-ligand interactions and corresponding conformational changes in the protein structures are of crucial importance in many biological functions, and in drug design. Although computer modelling has been used to predict binding affinities, the strengths of such interactions are normally determined by various experimental assays.

Affinity determination of antibody-peptide interactions is important for characterising recognition specificities of antibodies, and to delineate the antigenic determinant (epitope mapping), with linear or continuous epitopes.

Protein-ligand interactions can be studied by (i) equilibrium titrations, in which the equilibrium concentrations of the ligand and protein are measured, (ii) kinetic measurements, in which the on- and off-rate constants for the protein are measured at binding equilibrium and the ratio gives the equilibrium constant and (iii) stability measurements in which the changes in protein stability are followed during protein binding and the free energy difference between free (apo form) protein and ligand-bound protein is determined. Other techniques, such as calorimetry, radiolabeling, and spectroscopy require large sample amounts or specifically labelled proteins.

A new methodology using hydrogen/deuterium amide exchange (HDX) was applied for the first time to determine the binding affinity of anti-3-nitrotyrosine antibody to nitrated PCS peptides⁵ [172]. The method combines the previously established approaches of Protein- Ligand Interaction by Mass Spectrometry, Titration, and H/D EXchange (PLIMSTEX), such that there is a dilution can be used in conjection with HDX (named dPLIMSTEX) and the mass of the peptide ligand is the readout instead of the mass of the antibody. A main advandage of the dilution strategy is to minimize

5dPLIMSTEX approach was applied for the Tyr-nitrated peptides-anti-3-nitrotyrosine antibodies interaction systems during my visit in St. Louis (MO, USA), Washington University (DAAD Programme).

protein consumption in the measurement. Additionally, this method provides information on the minimum number of amino acids involved in the interaction [172].

Many factors such as pH, temperature, neighbouring side chains and isotope involvement can affect the proton transfer reactions between amides and solvent. The exchange rate of the hydrogens depends on the type of hydrogens (Figure 47): (i) amide hydrogens will exchange at a rate depending on their intra-molecular hydrogen bonding and access to the solvent; (ii) hydrogens on carbons undergo very slow H/D exchange; (iii) hydrogens on the amino terminus of certain amino acids chains (e.g.

aspartic acid, glutamic acid, lysine, serine) exchange very rapidly as compared to solvent e which exchanges slowly.

N CH C

Figure 47: Chemical structure of –Ala-Glu-Asn- peptide sequence pointing out three different types of hydrogens in proteins and their fate in the H/D exchange.

Blue H: hydrogens covalently bonded to carbon undergo extremely slow H/D exchange; green H: hydrogens bonded to nitrogen and oxygen in the side chains exchange extremely fast H/D exchange; bold red H: backbone amide hydrogens exchange in our time scale, sub-sec to many days.

The rates for individual amide hydrogen exchange are both pH dependent and temperature dependent. Amide hydrogen exchange can be either acid- or base-catalysed.

k_ex = k_H [H⁺] + k_OH [OH^-] ( 1 )

In equation (1), kH and kOH represent the rate constants for acid- and base-catalyzed reactions. For most peptides, acid- and base-catalyzed H/D exchange rates are

approximately equal when the pH is between 2 and 3, and the apparent rates are the slowest.

The HDX approach enables (i) the determination of dynamics nature of proteins in solution by determination of folding or unfolding rates [49]; (ii) calculation of association and dissociation constants; (iii) separation of different conformers and (iv) mapping of protein interfaces that become sterically protected. Additionally, it provides some advantages over other techniques such as low sample requirements (ca.

500 pmol) and no size limitation for the protein, as even large protein complexes can be studied.

Interaction studies by HDX are based on specific interactions between protein and ligand, as well as protection of the binding sites from HDX. The protection of the binding sites allows characterisation of the interaction by ESI-MS with identification of the exact number of amide bound hydrogens protected by the binding.

In the dPLIMSTEX method (Figure 48), antibody and peptide solutions were incubated for 2 h at 20 °C to establish equilibrium before starting the HDX experiment. To initiate the HD exchange the antibody-peptide complex solution or the peptide alone were mixed with an equal volume of D₂O buffer (with the same salt composition as in the aqueous solution) at 20 °C. After an established time for H/D exchange, the exchange was quenched by adding ice-cold HCl solution, producing a final pH of 2.5, and the sample was snap frozen in liquid nitrogen. After removal of the frozen sample from the liquid nitrogen, a zip-tip desalting procedure was performed with ice-cold solvents within 30-50 sec to minimize back-exchange. The desalted peptide solution was immediately injected into the mass spectrometer through a steel T-union which was connected with an isocratic LC flow of 50 % acetonitrile containing 0.1 % HCOOH at 40 µL min^-1. Both the T-union and the capillaries for the LC and mass spectrometer connections were placed in an ice bath.

Peptide Antibody +

20°C 2h

+ D₂O HCl till pH 2.5

Quench N₂liquid

Zip-tip preparation

Zip-tip preparation

Desalting the sample by zip-tip

LC –flow

ESI-MS Syringe

a

b

Figure 48: dPLIMSTEX method consists of two parts: (a) Complex formation between the antibody and the peptide, followed by H/D exchange and quenching the reaction in N₂ liquid; and (b) Desalting of the samples with a previous prepared Zip-tip column with 0 °C cold solution and analysis of the peptide by mass spectrometry [173].

The dPLIMSTEX method involves dilutions steps of the complex such that a half volume of the starting antibody-peptide solution was equilibrated directly to form the complex, and the other half volume was diluted with aqueous buffer before incubation.

The latter solution was further divided and diluted by the same procedure. The dilution steps could be continued until the concentration of the peptide became so low that it is was no longer detectable by the mass spectrometer (Figure 49).

Antibody x µM peptide, respectively, and z is the dilution factor [172, 173].

The dPLIMSTEX results were fitted by using the Mathcad program (Washington University). The model curves from dPLIMSTEX results are illustrated in Figure 50. This represents the theoretical calculation of dPLIMSTEX results using a series of theoretical curves based on a 1:1 binding model with K_a = 1 µM (association constant) and ∆D1 = 5 (ligand deuterium shifts upon binding, ∆Di, i = 1 to n, which is the difference between the average deuterated mass of the bound ligand in the complex and that of the apo-ligand form). The upper horizontal line represents the value of D₀, the free peptide mass after HDX determined without any antibody present and represents the average of replicate measurements.

Figure 50: Theoretical dPLIMSTEX curves generated from the modelling program implemented in Mathcad. The curves were calculated based on a 1:1 binding model with K_a = 1 µM and ∆D1 = 5. The curves correspond to a two-fold dilution series of different [Ab] values, where [Ab] refers to the total concentration of the antibody (i.e., each curve represents a 2-fold dilution from the curve immediately below it). When the [Peptide]/[Ab]

ratio is low, generally the dPLIMSTEX curves exhibit relatively large slopes and curvatures, as shown in the “steep region” The curves become flattened and their slopes become smaller in the “flat region”. The curves will eventually approach the upper horizontal line when the [Peptide]/[Ab]

ratio reaches infinity. Data distributed in the “steep region” are more sensitive to changes of binding affinity and dilution factor, thus carrying more information for determining fitting parameters (K_a and ∆D1). The data modelling results depend on the shape of the fitting curves and the mass difference between data points; thus, it is important to measure all data points under the same experimental conditions so that inevitable back exchange will be consistent for all the experiments.

The dPLIMSTEX method was applied here for the first time to peptide-antibody immune complexes with the characterisation of the recognition specificity of a monoclonal antibody to different Tyr-nitrated PCS peptides [173].

It has been shown that prostacyclin synthase (PCS) in bovine aortic microsomes undergoes specific nitration at Tyr-430 upon treatment with peroxynitrite, as identified by high resolution power mass spectrometry. The epitope recognition motif for the MAB5404 antibody includes nitrated Tyr as well basic amino acids surrounding the nitrated Tyrosine. dPLIMSTEX was applied to the anti-3-nitrotyrosine-PCS peptides interacting with two antibodies against 3-nitrotyrosine [173]. The characteristics of the antibodies are summarised in Table 5 and for the peptides in Table 7.

Table 7: PCS peptide sequences used in the dPLIMSTEX approach.

Code Peptide sequence Nitration site

1 H-⁴¹⁶EKKDFY⁴²¹(NO₂)KDGKRL⁴²⁶-OH Tyr-421 3 H-⁷⁹DPHSY⁸³(NO₂)DAVVWEPR⁹¹-OH Tyr-83 5 H-⁴²⁴GKRLKNY⁴³⁰(NO2)SLPWGA⁴²⁶-OH Tyr-430

2 H-⁴¹⁶EKKDFY⁴²¹KDGKRL⁴²⁶-OH -

6 H-⁴²⁴GKRLKNY⁴³⁰SLPWGA⁴²⁶-OH -

The Tyr-nitrated PCS peptide 3 was characterised by LTQ Orbitrap-MS (Figure 51) and doubly and triply charged ions were identified. The most intensive peak was chosen for analysis by HDX of the peptide alone, as well of the peptide bound to the 39B6 antibody.

400 600 800 1000 1200 1400 1600 1800 2000

539.0 539.5 540.0 540.5 541.0 541.5 542.0 542.5

539.2446

Figure 51: ESI mass spectrum of Tyr-nitrated PCS peptide 3 showing the protonated doubly and triply charged ions. A zoom of the monoisotopic distribution of triply charged ions is present in the right part.

Solutions of the anti-3NT-antibody and a series of different concentrations of the Tyr-nitrated PCS peptide 3 were prepared in a buffer solution (150 mM KCl and 10 mM HEPES, pH 7.4). For each sample tested, an antibody solution was mixed with an equal volume of PCS peptide 3 and incubated for 2 h at 20 °C, to establish equilibrium before starting the HD exchange. To initiate the exchange, a solution of antibody-peptide complex or PCS peptide 3 alone was mixed with an equal volume of D₂O /HEPES buffer (with the same salt composition as in the aqueous solution) at 20

°C. After 65 sec, the exchange was quenched by adding ice-colded HCl solution producing a final pH of approx 2.5. The sample was quickly frozen in liquid nitrogen and kept frozen until desalting and further analysis. C18 zip tips were used to desalt the antigenic peptide. The frozen sample was removed from liquid nitrogen and slightly hand-warmed. While half of the sample volume was melted and the other half remained frozen, the melted portion was introduced to the zip tip washed using 0.1 % HCOOH and eluted using 50 % ACN/0.1 % HCOOH. The zip tip procedure was

through a steel T-union into the mass spectrometer. The T-union used was connected directly to the mass spectrometer with an isocratic flow of 50 % acetonitrile containing 0.1 % formic acid at flow rate of 40 µL min^-1. The T-union, LC capillaries and all mass spectrometer connections were placed in an ice bath. For each concentration, duplicate or triplicate experiments under the same experimental conditions were conducted, and mass spectra were acquired in the positive-ion mode and a zoom-scan in the mass range, m/z 539-546. The mass spectrum of the Tyr-nitrated PCS peptide 3 after HDX showed a centroid at m/z 541.2608 (Figure 52) and the centroid of the monoisotopic distribution was calculated using equation 2:

Abundance)

539 540 541 542 543 544 545

m/z

Figure 52: ESI-MS spectrum (LTQ Orbitrap XL-MS) of Tyr-nitrated PCS peptide 3 after HDX. The centroid of monoisotopic distribution of Tyr-nitrated PCS peptide 3 was established at m/z 541.2608.

The centroid of the monoisotopic distribution of Tyr-nitrated PCS peptide 3 was established at m/z 539.5531 (Figure 53) and at 541.2608 after HDX. This result

indicated that 11 hydrogens of the amides in the Tyr-nitrated PCS peptide 3 were exchanged by deuterium (Figure 53). The exchange of 11 hydsrogens were calculated using the equations (3), (4) and (5):

MW (HDX PCS peptide 3) = m/z (HDX PCS peptide3) x 3 – 3 x MW(H⁺)= A ( 3 )

MW (PCS peptide 3) = m/z ( PCS peptide 3) x 3 – 3 x MW(H⁺)= B ( 4 )

11 = (A-B) x 2 ( 5 )

539.00 539.50 540.00 540.50 541.00 541.50 542.00 542.50 543.00 543.50 544.00

539.5531 541.2608 PCS peptide

3

HDX PCS peptide

3

3 : H-⁷⁹DPHSY⁸³(NO₂)DAVVWEPR⁹¹-OH

Figure 53: ESI-MS spectrum of PCS peptide 3 and the HDX of free PCS peptide 3.

The centroid of both mass spectra was calculated and the overlay plot is presented hierhin.

Typically, high affinity antibody-antigen interactions have K_D values in the range of 10^-8-10^-10 M [174]. Considering the detection limit of current mass spectrometers in the ESI mode, the lower concentration limits of antibody and antigenic peptide were

For 39B6 antibody-Tyr-nitrated PCS peptide 3 interaction, the lowest concentrations applied were 41 and 20.5 nM, respectively. The 39B6 antibody was diluted to concentrations of the Fab part of the antibody of 660 nM, 330 nM, 165 nM, 82 nM and 41 nM, and these were mixed the Tyr-nitrated peptide 3 at ratios of 1:6, 1:2 and 1:1 for the dPLIMSTEX analysis. The centroid of all mass spectra obtained from all Fab concentrations and Fab:Tyr-nitrated PCS peptide 3 ratios were calculated by Excel, and a 1:1 binding model was used for the curve fitting.

Figure 54 shows the overlapping of ESI-mass spectrum of HD exchange of free and antibody bound Tyr-nitrated PCS peptide to peptide 3. A small offset of 0.0544 was obtained between the the centroid of the two spectra.

539 539.5 540 540.5 541 541.5 542 542.5 543

541.2064 541.2608

Figure 54: Overlapping of ESI mass spectrum of HDX of free (blue) and bound Tyr-nitrated PCS peptide 3 (red). A small offset of 0.0544 was obtained between the the centroid of the two spectra.

The fitting result corresponding to the Tyr-nitrated peptide-antibody complex is presented in Figure 55. Different ratios of Fab (39B6 antibody)-PCS peptide of 1:6, 1:2 and 1:1 and Fab concentrations of 660 nM, 330 nM, 165 nM, 82 nM and 41 nM were used in the dPLIMSTEX experiment.

[3] : [Fab39B6 antibody]

1618 1618,5 1619 1619,5 1620 1620,5

0 5 10 15 20

a

b

D₀

[3] : [Fab39B6 antibody]

1617,75 1618,25 1618,75 1619,25 1619,75

0 0,5 1 1,5 2 2,5 3

Peptide massPeptide mass

Figure 55: (a) dPLIMSTEX fitting curves for 39B6 antibody-PCS peptide 3 interactions. The concentration of antibody Fab regions (shown as [Fab 39B6 antibody]) is used in place of the antibody concentration in the modelling. [Fab39B6 antibody] for the dashed line is 660 nM, whereas [Fab_39B6

antibody] for the solid lines 41nM. (b) The curve fitting is zoomed in for better illustration of the steep region and the separation of the two fitted curves. The D₀ mass is for the unbound peptide and is depicted as a line representing the asymptote for the other curves. Note that in the analysis and the plot Fab concentration was used and not the antibody concentration [173].

The two fitted curves in Figure 55 represent a 16-fold dilution from the lower dashed

most likely because the lowest antibody concentration is still one magnitude higher than the KD value. In the “steep region” more data points were collected in order to define better the curve shape and to afford higher modelling sensitivity. A two-fold dilution series was not appropriate as the data were almost overlapping and the resulting curves lacked were unable to provide a good fit.

From the fit of the 39B6 antibody:PCS peptide 3 interaction shown in Figure 55, a K_D value of 3nM was determined. The root mean square (RMS) for the modeling of 0.094, representing the deviation between fit and experimental data, was insignificant, thus substantiating the reliability of the fitting results. In addition, the binding stoichiometry was assessed by the dPLIMSTEX approach. IgG antibodies may bind two antigens, each Fab region interacting with one antigen. However, a 1:1 antibody-antigen binding stoichiometry was determined. The dashed fitted curve in Figure 55, obtained at an antibody concentration of more than two orders of magnitude larger than K_D, is almost a “sharp-break” curve, from which the binding stoichiometry is obtained; this break occurs at [3]:[Fab(39B6 antibody)] = 1. Importantly, the Fab concentration not the antibody concentration was used in this calculation. At the standard case of two Fab entities per antibody, the antibody-peptide complex would yield a 1:2-binding model system. The result providing a 1:1 stoichiometry also suggests that the two binding events per antibody are independent. Furthermore, the modeling of this antibody-peptide suggests at least 5 backbone amide protons from the antigenic peptides are sequestered from HDX upon binding. This result further suggests that at least five amino acids residues constitute the epitope.

In general there is a competition in the HD exchange of the peptide versus peptide- antibody complex, depending on the exchange rate constant of the peptide. In the 39B6 antibody-peptide 3 complex, the exchange rate constant of the peptide 3 is higher as dissociation rate constant of the 39B6 antibody-peptide 3 complex (Figure 56).

Antibody-Peptide _HHHH

Antibody-Peptide_DDDD

Antibody + Peptide _HHHH

Peptide_DDDD k_off

k_on

k_ex

Figure 56: General scheme of the HDX during complex formation between two molecules, in this case an antibody and a peptide.

The HDX kinetics curve of peptide 3 was treated with two-group modelling and an average chemical exchange rate constant k_ex of free peptide 3 was determined at 0.69 sec^-1 for 58 % exchangeable amide protons and 0.077 sec^-1 for the remaining 42 % exchangeable amide protons (Figure 57). Compared to the association rate k_on of the 39B6 antibody- peptide 3 complex, k_ex >> k_on. Therefore, the backbone amide hydrogens in the peptide likely undergo a correlated exchange owing to the relatively slow association rate (i.e., HDX occurs during the time following dissociation of the complex and before the peptide can form a new complex) [175]. This can be considered as an inevitable “leak” or loss of measured protected amide sites in the

∆D1 (ligand deuterium shifts upon binding, ∆Dⁱ (i = 1 to n) which is the difference between the average deuterated mass of the bound ligand in the complex and that of the apo-ligand form). Consequently, the actual number of amide protons that becomes sequestered upon complex formation is larger than that calculated from ∆D1.

Figure 57: The kinetic curve for H/D exchange of free PCS peptide 3. All the data of deuterium uptake was normalised to the highest average uptake value. The error bars shown for the data points represent the standard deviation from duplicate independent experiments.

To clarify further the molecular recognition properties and specificity of the two monoclonal anti-nitrotyrosine antibodies (Table 5), the MAB5404 and 39B6, the binding affinities of the antibody-complexes with three nitrated peptides containing different tyrosine nitration sites, and two peptides with no nitration sites were determined (Table 7). The results showed strong binding for peptides 1, 3 and 5 with both MAB5404 and 39B6 antibodies, whith dissociation constants in the low nM range (Table 8). These results showed specific recognition for nitrotyrosine by both antibodies. The modeling procedure [173] is based on the experimental data with Fab concentration (Fab concentration, which is equal to twice the antibody concentration) of 41 nM.

Table 8: The dPLIMSTEX results of the MAB5404 antibody and 39B6 antibody with five PCS peptides determined from the data modeling for K_D and ∆D1.

MAB5404 antibody 39B6 antibody Code Peptide sequence

K_D[M] ∆D K_D[M] ∆D

1 H-⁴¹⁶EKKDFY⁴²¹(NO₂)KDGKRL⁴²⁶-OH (2.9 ± 0.9) E-9 0.56 ± 0.03 (7.9 ± 1.6) E-9 0.55 ± 0.05 3 H-⁷⁹DPHSY⁸³(NO₂)DAVVWEPR⁹¹-OH (18.1 ±0.8) E-9 1.80 ± 0.30 (3 ± 0.3) E-9 2.3 ± 0.1 5 H-⁴²⁴GKRLKNY⁴³⁰(NO₂)SLPWGA⁴²⁶-OH (13.8 ±3.4) E-9 2.40 ± 0.20 (11.4 ± 2.4) E-9 1.7 ± 0.1 2 H-⁴¹⁶EKKDFY⁴²¹KDGKRL⁴²⁶-OH (6 ±0.8) E-6 - (0.5 ± 0.3) E-3 - 6 H-⁴²⁴GKRLKNY⁴³⁰SLPWGA⁴²⁶-OH (2 ±0.8) E-6 - (0.7 ± 0.2) E-3 -

Note that the Fab concentration was used in the determination of the KD values of these peptide-antibodies interactions systems and not the antibody concentration.

The fitted curves in Figure 58 have a steeper region (with higher slope) at a 0-5 peptide-Fab ration concentration, where the shape of the curves is more sensitive to the changes of unknown influences. The slopes become smaller and the curves flatter at peptide-Fab ration concentration > 5. To better define the unknown influences from the modeling and to generate more reliable fitted curves, more data points were collected in the steeper region. Interestingly, the affinities of MAB5404 antibody with peptides 1 and 5 (> 10⁸) are slightly larger than for peptide 3. It is important to note that the peptides 1 and 5 contain multiple basic amino acids residues, Lys and Arg, surrounding a nitrotyrosine residue. The K_D values for the MAB5404 antibody bound to PCS peptides 1, 3 and 5 are in agreement with the epitope identified. MAB5404 antibody bound specifically to macromolecules which contains nitrated Tyr surrounded by positively charged residues (Lys, Arg) [141].

In contrast to the MAB5404 antibody, a similar affinity dependence of the Tyr-nitrated peptides was not obtained for the 39B6 antibody. The 39B6 antibody binds to PCS peptide 3 with a 2-3 fold higher binding constant than that of peptide 1 and 5. One possible explanation for this result may be that the paratope of the 39B6 antibody comprises addition hydrophobic interactions with the peptides, such as PCS peptide 3 containing four continuous hydrophobic residues at the C-terminal side of the

In contrast to the MAB5404 antibody, a similar affinity dependence of the Tyr-nitrated peptides was not obtained for the 39B6 antibody. The 39B6 antibody binds to PCS peptide 3 with a 2-3 fold higher binding constant than that of peptide 1 and 5. One possible explanation for this result may be that the paratope of the 39B6 antibody comprises addition hydrophobic interactions with the peptides, such as PCS peptide 3 containing four continuous hydrophobic residues at the C-terminal side of the

Im Dokument Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions (Seite 88-109)