Exp Mol Med.  2007 Feb;39(1):74-83.

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

Affiliations
  • 1Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA. gaillard@niu.edu
  • 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY, USA.
  • 3Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY, USA.

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.

Keyword

3-nitrotyrosine; Bruch membrane; collagen type IV; inflammation; nitrites

MeSH Terms

Tyrosine/*metabolism
Tandem Mass Spectrometry
Substrate Specificity
Nitrites/*metabolism
Humans
Collagen Type IV/*metabolism
Chromatography, Liquid
Binding Sites
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