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D.C. Paik, Z. Wang, E.R. Gaillard; Sites of Tyrosine Nitration Identified by LC/MS in Nitrite Modified Collagen Type IV: Implications to Retinal Pigment Epithelial Cell Dysfunction . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5292.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Reactions involving nitrite ion are becoming increasingly recognized as a means through which oxides of nitrogen (including nitric oxide and nitrogen dioxide) exert negative effects on human tissues. 3–nitro–tyrosine (3NT) is an important biomarker of nitrosative damage and has been detected in several human age–related diseases. We have recently shown that nitrite modification of basement membrane proteins, including meshwork collagen type IV, has deleterious effects on overlying retinal pigment epithelial (RPE) cell viability (Wang et al, in press). The present study was undertaken in order to confirm the formation of 3NT by reaction of nitrite with purified collagen type IV and to identify specific tyrosine residues that may be modified. Methods: Collagen type IV was modified via incubation with 200 mM NaNO2 (pH = 7.38) for one week at 37oC. Complete removal of unreacted nitrite was confirmed by colorimetric Greiss assay. The modified protein was digested with a protease cocktail (collagenase, pronase, pepsin and trypsin) and the liberated 3–NT was detected via LC/MS (ThermoElectron, LCQ Advantage, Surveyor, electrospray ionization, Synergi Polar C12 RP column). LC mobile phase was ACN balanced with H2O (0.025% acetic acid) in the following gradients: 0–2% ACN for 15mins, 2–25% ACN for 35 mins, 25–95% ACN for 70mins and a flow rate 0.3ml/min. For sequence analysis, the modified collagen was subjected to trypsin digestion only. After digestion, the fraction containing MW<10,000 was concentrated and analyzed by LC/MS. MS/MS and SEQUEST analyses were used to determine sites of nitration. Results: Under the present chromatographic conditions, 3NT was detected in protein digests of nitrite modified collagen IV with a Rt=24.5min. Positive identification was confirmed by identical Rt, max = 279 nm and 355 nm, and m/z = 227 with standard 3NT, and was not detectable in non–modified collagen. In addition, analyses of tryptic digests indentified four sites of tyrosine nitration in the α1 chain and one site in the α2 chain. These sites are located in the triple–helical region of the protein. Conclusions: 3–nitro–tyrosine is an important biomarker of nitrosative damage. Our study indicates that tyrosine nitration in collagen type IV occurs by reaction with nitrite at neutral pH and is located in the helical region. These sites represent possible ligands in RPE cell–matrix interactions.
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