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KMID : 0620920070390010074
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Experimental & Molecular Medicine
2007 Volume.39 No. 1 p.74 ~ p.83
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Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV
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Wang Zhen
Paik David C.
Dillon James P.
Gaillard Elizabeth R.
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Abstract
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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 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 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 Rt, lmax=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, a1(IV)Y348, a1(IV)Y534, a2(IV)Y327, and a2(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.
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KEYWORD
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3-nitrotyrosine Bruch membrane, collagen type IV, inflammation, nitrites
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