Tyrosine nitration site specificity identified by LC/MS innitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-link-ing results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently ob-served that nitrite modification of basement mem-brane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement mem-branes. Human collagen type IV was modified via incubation with 200 mM NaNO2 (pH = 7.38) for one week at 37oC. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C12 RP column. Site specificity was determined from tan-dem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical Rt, λmax= 279 nm and 355 nm, and m/z= 227. Analyses of tryptic digests identified four sites of tyrosine ni-tration, α1(IV)Y348, α1(IV)Y534, α2(IV)Y327, and α2(IV)-Y1081. These sites are located in the triple- helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV. KCI Citation Count: 8