title: Specific enrichment of a targeted nitrotyrosine-containing peptide from complex matrices and relative quantification for liquid chromatography-mass spectrometry analysis.
abstract: Protein tyrosine nitration is considered an important non-enzymatic post-translational modification. In the tyrosine nitration process, 3-nitrotyrosine is formed and recognized as a biomarker of nitrosative/nitrative stress implicated in inflammatory responses and age-related disorders. In view of the complexity of biological samples and the ultra-low abundance of protein-incorporated nitrotyrosine, selective enrichment of nitrotyrosine-containing peptides prior to chromatographic separation is crucial. Herein, I report a simple yet highly specific and efficient enrichment method for nitrotyrosine-containing peptides. After blocking all primary amines in the sample by acetylation with acetic anhydride, I then further converted all nitrotyrosine residues into aminotyrosine residues by reduction with dithiothreitol and hemin. Therefore, I eliminated the side-product with 80Da adduct, since inevitable considerable amount of which was generated in the widely used reduction mediated by sodium dithionite. Both acetylation and reduction yields were close to 100%, and my one-pot sample derivatization applied no solid phase extraction steps or sample transference to avoid sample loss. To capture and release aminotyrosine-containing peptides, I synthesized an N-hydroxysuccinimide-ester-functionalized stationary phase which had very high affinity towards amino groups and possessed a base-cleavable ester linker to retrieve targeted peptides by hydrolysis. I validated this strategy by highly efficient enrichment of the targeted peptide from complex matrices of trypsin-digested bovine serum albumin (BSA) and human plasma spiked with derivatized nitrotyrosine-containing angiotensin II. My enrichment method successfully removed most untargeted peptides in those samples. By relative quantification with home-made identical and stable-isotope labelled internal standards, I investigated the recoveries of a nitrotyrosine-containing peptide from complex biological matrices during enrichment for the first time. Mean recoveries were 49.8% and 41.1% (n=6) for the enrichment of nitrotyrosine-containing angiotensin II from 1:100 (w/w) BSA digest and from 1:10 000 (w/w) human plasma digest, respectively. My enrichment method demonstrated great potential in future applications to clinical samples and biomarker discovery.
authors.affiliation: College of Chemistry & Materials Science, Northwest University, Xi'an 710069, PR China. Electronic address: yangyunwww@126.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000107:Acetylation; D000804:Angiotensin II / Q000097:blood / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D009574:Nitro Compounds / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D009574:Nitro Compounds; D010455:Peptides; D000804:Angiotensin II; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine
keywords: Aminotyrosine; Enrichment; LC–MS; Nitrotyrosine; Relative quantification