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Petre B.-A.,Laboratory of Analytical Chemistry and Biopolymer Structure Analysis and Steinbeis Research | Petre B.-A.,Al. I. Cuza University | Ulrich M.,University Hospital of Tuebingen | Stumbaum M.,Laboratory of Analytical Chemistry and Biopolymer Structure Analysis and Steinbeis Research | And 4 more authors.
Journal of the American Society for Mass Spectrometry | Year: 2012

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT-residue, located at specific surface-accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity-MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinitymass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations. © American Society for Mass Spectrometry, 2012.

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