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

• Published in: Experimental & molecular medicine Vol. 39; no. 1; pp. 74 - 83
• Main Authors: Wang, Zhen, Paik, David C, Dillon, James P, Gaillard, Elizabeth R
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 28-02-2007
• Subjects: Binding Sites; Chromatography, Liquid; Collagen Type IV - metabolism; Humans; Nitrites - metabolism; Substrate Specificity; Tandem Mass Spectrometry; Tyrosine - metabolism
• ISSN: 1226-3613; 2092-6413; 2092-6413
• DOI: 10.1038/emm.2007.9

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.