Affinity-mass spectrometric characterization of anti-3-nitro tyrosine antibody

This methodology has been developed primarily in our laboratory for identification of specific epitopes of antibody-bound proteins based on the determination of B-cell epitope sequences as a result of the observation, that an antibody will protect the binding site(s) of a bound peptide or protein antigen from proteolytic cleavage [220]. In the present, study the procedure was employed for identification and mass spectrometric characterization of oxidative modified proteins.

Initially several experiments were performed using nitrated and non-nitrated synthetic peptides. The formation of antigen-antibody complexes has been employed for the development of a general approach for the identification of protein epitopes by mass spectrometry, by combining limited proteolytic cleavage of intact immune complexes (epitope excision) with mass spectrometric peptide mapping [221, 222]. The initial applications of mass spectrometric epitope mapping have been successfully applied on small sequence epitopes such as from purified polypeptides [223]. Recent studies have shown that large, native proteins including conformational epitopes [224], can be determined very accurately by mass spectrometry [225]. These molecular antigen-antibody interaction features have led to the well-established mass spectrometric epitope extraction/epitope excision procedure. The epitope excision procedure consists in immobilizing the antibody on a micro-column and binding of the antigen molecule to this column. The proteolytic digestion using specific proteases is then carried out, the non-bound fragments are washed away and the epitope-antibody complex is dissociated followed by analysis using mass spectrometry. In the epitope extraction procedure, the antigen molecule is first proteolytically digested and then applied on to the antibody column [21]. The general analytical concept of the mass spectrometric epitope analysis approach is summarised in Figure 2.54.

Y

Washing until no MS signal Elution with 0.1% TFA Binding nitrated

Washing until no MS signal Elution with 0.1% TFA Binding nitrated

Figure 2.54. The principle of affinity-mass spectrometry experiments using immobilized antibodies.

The antigen is digested in solution, and then the resulting peptide mixture is subjected to the Sepharose – immobilized antibody column. Washing steps are performed to remove the non-binding peptide fragments; the remaining affinity-bound peptide(s) are eluted and analysed by mass spectrometry.

Anti-3-nitro tyrosine antibody (MAB5404) was immobilized on sepharose in a proteolytic affinity extraction-MS approach employed for the affinity enrichment of oxidative modified proteins from cystic fibrosis sputum sample. The principle is analogous to the epitope extraction-MS method, where the protein containing the antigen motif is digested in solution and the proteolytic peptide mixture is added to the antibody column. The immune complex is allowed to be formed and the peptides remaining in solution are removed and collected as non-binding fraction. Due to the high antibody - antigen specificity, only peptides containing the antigenic determinant (3-nitro tyrosine) will interact with the paratope; the corresponding antibody sequence responsible for interaction with the epitope. In the second step, the matrix is washed extensively in order to ensure complete removal of unbound peptides. The last volume of the washing buffer, referred to as washing fraction is collected and analyzed by MS. The peptides remaining bound to the antibody are eluted (elution fraction) under different conditions using 0.1% trifluoroacetic acid (TFA). The antibody columns were prepared by coupling the mouse antibody MAB5404 to NHS-activated Sepharose, as described in Experimental part. The PCS peptides were dissolved in PBS buffer, pH 7.4 and added to the antibody columns. After 2 hrs incubation, the unbound peptides were removed, the column was washed several times with PBS buffer, and the peptide-antibody complexes were dissociated using

0.1 % TFA. The first washing fractions containing unbound peptides, and the elution fractions, which contain the peptides specifically bound to the anti-3-nitro tyrosine antibody column were collected, lyophilized and analysed by mass spectrometry. As Figure 2.55 shows only the nitrated PCS peptides were found in the elution fractions.

1747.23

1793.77 1777.12

DFYKDGKRLKN -OH [M+H]⁺: 1791.90 DFYKDGKRLKNYSL-OH [M+H]⁺: 1745.91

Y(NO₂)SL

1792.74 1776.34 1759.97

Figure 2.55. Affinity-mass spectrometry analysis using nitrated and non-nitrated PCS (419-432).

Peptides were inculated for 2 h at 25^oC with the monoclonal antibody MAB5404 immobilized on NHS-activated sepharose; MALDI-TOF-MS shows the presence of both nitrated and non-nitrated peptides in the supernatant fractions, while in the elution fractions was identified only the nitrated PCS meaning that the antibody was successfully bound on the clolumn

Nitrated PCS peptides underwent photochemical fragmentation under UV-MALDI MS, which has led to the formation of 3-nitroso-tyrosine derivative [Tyr (NO)], by the loss of one oxygen and the formation of nitrene type fragment [Tyr (:N:)] by the loss of two oxygens and also the reduction of nitro-group to form an amine [Tyr (NH2)] [226]. Additionally, similar affinity mass spectrometric experiments were performed, but the measurement of supernatant and elution fractions was carried out by ESI - MS instrument obtaining the same results as in the MALDI-TOF experiments Figure 2.56.

DFYKDGKRLKN -OH [M+H]⁺: 1791.90 DFYKDGKRLKNYSL-OH [M+H]⁺: 1745.91

285.0

200 400 600 800 1000 1200 1400 m/z

Supernatant

200 400 600 800 1000 1200 1400 m/z

Elution

DFYKDGKRLKN -OH [M+H]⁺: 1791.90 DFYKDGKRLKNYSL-OH [M+H]⁺: 1745.91

200 400 600 800 1000 1200 1400 m/z

Supernatant

200 400 600 800 1000 1200 1400 m/z

Elution

DFYKDGKRLKN -OH [M+H]⁺: 1791.90 DFYKDGKRLKNYSL-OH [M+H]⁺: 1745.91

Y(NO₂)SL

[M+3H]³⁺

[M+4H]⁴⁺

[M+2H]²⁺

Figure 2.56. ESI - MS analysis of affinity experiments using nitrated and non-nitrated PCS (419-432) peptides were inculated on the monoclonal antibody MAB5404 affinity column. Supernatant and elution fractions were measured by direct analysis using ESI-MS. The peptides fraction were concentrated in the guard column and, then analysed by MS confirming the results obtained by MALDI-TOF MS analysis

2.6.5 Mass spectrometric identification of oxidatively modified proteins