Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): analytical challenges for the identification of modified sites.
OBJECTIVE
We examined the metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF) using the Cu(II)/ascorbate/O2 model oxidative system.


METHODS
Electrospray ionization mass spectrometry, peptide mapping and amino acid analysis were utilized to determine the nature of the covalent modification induced by the metal-catalyzed oxidative system. Additionally, analytical ultracentrifugation, the Bradford assay, circular dichroism and ANSA dye-binding were used to determine the nature of any conformational changes induced by the oxidation.


RESULTS
Exposure of BDNF to the Cu(II)/ascorbate/O2 system led to the modification of ca. 35% of Met92 to its sulfoxide, and to subsequent conformational changes. The proteolytic digestion procedure was sensitive to this conformational change, and was unable to detect the modification. Chemical digestion with CNBr, however, was not sensitive to this change, and allowed for the identification of the site of modification.


CONCLUSIONS
The modification of Met92 to its sulfoxide rendered the oxidized BDNF inaccessible to proteolytic digestion, due to conformational changes associated with the oxidation.
Jensen
J L
JL
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
Kolvenbach
C
C
Roy
S
S
Schöneich
C
C
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000737:chemistry; D010450:Endopeptidases / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation
D000817:Anilino Naphthalenesulfonates; D019208:Brain-Derived Neurotrophic Factor; D005456:Fluorescent Dyes; D003300:Copper; C039256:6-anilino-1-naphthalenesulfonic acid; D008715:Methionine; D010450:Endopeptidases; D003488:Cyanogen Bromide; D001205:Ascorbic Acid; D010100:Oxygen; C013111:methionine sulfoxide