Identification of nitration sites on surfactant protein A by tandem electrospray mass spectrometry.
Previous studies have shown that exposure of human surfactant protein A (SP-A) to nitrating agents [peroxynitrite (ONOO-); tetranitromethane (TNM; pH 8)] leads to nitrotyrosine formation. However, specific sites of nitration have not been identified. Herein, human SP-A, dissolved in Hepes buffer, was incubated with two boluses each of 0.5 mM ONOO- (pH 7.4) or 0.5 mM TNM (pH 8.0) for 15 min. After 30 min, SP-A samples were reduced, alkylated, and trypsin digested. The nitrated peptides and sites of amino acid nitration on the protein were identified by capillary high-performance liquid chromatography-coupled electrospray ionization tandem mass spectrometry (LC-ESMS/MS). The major nitrated peptide on both TNM- and (ONOO-)-exposed SP-A was the tryptic fragment Tyr161-Arg179 (YNTYAYVGLTEGPSPGDFR), located in the SP-A carbohydrate recognition domain. Sequencing of this nitrated peptide by LC-ESMS/MS demonstrated that the nitration was equally distributed on Tyr164 and Tyr166. A second lesser nitrated peptide corresponding to tryptic fragment Asn217-Arg222 (NCLYSR) was also found on TNM- and (ONOO-)-modified SP-A. No other nitrated amino acid was detected. Nitrated SP-A exhibited decreased ability to aggregate surfactant lipids in the presence of Ca2+. These data demonstrate that nitration of a specific tyrosine decreased an important protein function.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35233-6810, USA.
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Greis
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Zhu
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Matalon
D000595:Amino Acid Sequence; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009566:Nitrates; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011510:Proteolipids / Q000737:chemistry*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D009566:Nitrates; D010446:Peptide Fragments; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C002744:3-nitrotyrosine; D014443:Tyrosine