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

Abstract

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 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 membranes. Human collagen type IV was modified via incubation with 200 mM NaNO2 (pH = 7.38) for one week at 37°C. 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 tandem 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 R t, λmax = 279 nm and 355 nm, and m/z = 227. Analyses of tryptic digests identified four sites of tyrosine nitration, 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.