Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV.
- Author:
Zhen WANG
1
;
David C PAIK
;
James P DILLON
;
Elizabeth R GAILLARD
Author Information
1. Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA. gaillard@niu.edu
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
3-nitrotyrosine;
Bruch membrane;
collagen type IV;
inflammation;
nitrites
- MeSH:
Tyrosine/*metabolism;
Tandem Mass Spectrometry;
Substrate Specificity;
Nitrites/*metabolism;
Humans;
Collagen Type IV/*metabolism;
Chromatography, Liquid;
Binding Sites
- From:Experimental & Molecular Medicine
2007;39(1):74-83
- CountryRepublic of Korea
- Language:English
-
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 37degrees 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 Rt, 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.
