title: 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 NaNO(2) (pH=7.38) for one week at 37(o)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 C(12) 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), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(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.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: David C
authors.initials: DC
authors.lastname: Paik
authors.forename: James P
authors.initials: JP
authors.lastname: Dillon
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Gaillard
mesh_terms: D001665:Binding Sites; D002853:Chromatography, Liquid; D024141:Collagen Type IV / Q000378:metabolism*; D006801:Humans; D009573:Nitrites / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D024141:Collagen Type IV; D009573:Nitrites; D014443:Tyrosine