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

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 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 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, λmax = 279 nm and 355 nm, and m/z = 227. Analyses of tryptic digests identified four sites of tyrosine nitration, α1(IV)Y348, α1(IV)Y534, α2(IV)Y327, and α2(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.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…For the ECP and EDN proteins, the identification of analogous specific Tyr-nitration sites is in agreement with the high degree of structural homology for both proteins [37]. These results demonstrate specific tyrosine nitrations in Eosinophil proteins and illustrate the proteolytic affinityextraction-MS as a highly efficient approach, far more specific and sensitive compared with LC-MS/MS approaches employed in previous studies [26,27] and in the identification of the self-nitration of eosinophil peroxidase (EPO) [38]. Moreover, the proteolytic affinity-extraction-MS of producing and submitting a peptide mixture is applicable to single proteins, protein complexes from biological material and protein bands from electrophoretic separations with comparable efficiency [28,36].…”

“…The affinity of the nitrated ECP peptide was found to be completely abolished upon replacing the positively charged residues Arg 28 , Arg 34 , Arg 36 , and Lys 38 by alanine, thus confirming the importance of cationic amino acids in the vicinity to the nitration site for affinity binding. Affinity determinations of the nitrated peptide in comparison to the intact ECP protein using the SAW biosensor provided dissociation constants of approximately 6 nM for the nitrated peptide ECP (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41), and 28 nM for the intact ECP.…”

“…Thus, UV-MALDI-MS has been found to be critical for unequivocal identification of nitrations from biological samples due to the photochemical decomposition of nitrotyrosine residues [24,25]. In contrast, electrospray ionization mass spectrometry and IR-MALDI-MS have been shown to provide unambiguous identifications of NT-modified peptides [25][26][27]. In previous studies, different approaches of MS have been successfully employed for identifying tyrosine nitrations upon reaction with peroxynitrite in vitro [12,28] and in vivo [3].…”

Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions

“…The progression of the disease is multifactorial with both genetic and environmental components. Among these are oxidative and nitrosative stress associated with smoking, light exposure/phototoxicity, and impaired phagocytosis . Epigenetic modifications have also been implicated in AMD pathophysiology …”

“…Among these are oxidative and nitrosative stress associated with smoking, light exposure/phototoxicity, and impaired phagocytosis. [4][5][6][7][8][9][10][11] Epigenetic modifications have also been implicated in AMD pathophysiology. [12][13][14][15][16] Aging causes morphological changes in Bruch's membrane, including calcification and fragmentation due to the sub-RPE deposits of drusen that are associated with macular degeneration.…”