Use of a nitrated protein or peptide sequence for the implementation of a method of diagnosis

Method of in vitro diagnosis of the state of severity and progressiveness of a chronic pathology associated with nitrating stress in a biological sample from an individual, comprising:

• quantitative assay, in a biological sample from an individual, of the degree of nitration of a first particular nitrated protein or physiological peptide sequence,
• quantification of said degree of nitration of said first particular protein or physiological peptide sequence by comparing the concentration of said first particular nitrated protein or physiological peptide sequence with the total concentration of said first particular protein or physiological peptide sequence, nitrated and non-nitrated, obtained from a biological sample from the same individual,
• comparing said degree of nitration of said first particular nitrated protein or physiological peptide sequence with the degree of nitration of a second particular protein or physiological peptide sequence obtained from a biological sample from an individual not affected by said chronic or acute pathology,
• said second particular protein or physiological peptide sequence being a variant or an isoform, or having an amino acid sequence identity of at least 90%, and in particular 100%, with the aforesaid first particular protein or physiological peptide sequence or also derived from the same wild-type or mutated gene,
• said first particular protein or physiological peptide sequence being nitrated on at least one tyrosine residue located in an equivalent position in the aforesaid second particular protein or physiological peptide sequence,
• deduction, from the comparison carried out in the preceding stage, of the degree of nitrating stress of the individual, which may correspond to a state of severity and progressiveness of said chronic or acute pathology.

Method of in vitro preparation of a particular nitrated protein or peptide sequence on tyrosine residues in which the nitrated tyrosine residues correspond to the physiologically nitrated tyrosine residues of a particular nitrated protein or physiological peptide sequence in chronic or acute pathologies associated with nitrating stress,

• said particular nitrated protein or peptide sequence having an amino acid sequence identity of at least 90%, and in particular 100%, with the aforesaid particular nitrated physiological protein, even gene comprising:
• in vitro nitration of a particular protein or peptide sequence by tetranitromethane in a molar ratio to the nitratable tyrosines of the particular protein or peptide sequence not exceeding 20:1, in an aqueous buffer with pH between 7.5 and 8.5,
• in vitro identification of the nitrated tyrosines of said particular nitrated protein, in particular by mass spectrometry, and
• optionally, comparing the nitrated tyrosines identified on said particular protein or peptide sequence with the tyrosine residues of the particular nitrated protein or physiological peptide sequence.