Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV
Experimental & Molecular Medicine
; : 74-83, 2007.
Article
em En
| WPRIM
| ID: wpr-37553
Biblioteca responsável:
WPRO
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.
Palavras-chave
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Especificidade por Substrato
/
Tirosina
/
Sítios de Ligação
/
Cromatografia Líquida
/
Colágeno Tipo IV
/
Espectrometria de Massas em Tandem
/
Nitritos
Limite:
Humans
Idioma:
En
Revista:
Experimental & Molecular Medicine
Ano de publicação:
2007
Tipo de documento:
Article