Fig. 8.

Bromination site in the α2NC1 domain of collagen IV from mouse kidney and its dependence on peroxidasin expression. NC1 domains of collagen IV were isolated from WT and Pxdn^−/− mice and analyzed by LC-MS/MS as described in Materials and Methods. (A and B) Evidence for bromination of Tyr¹⁴⁸⁵ in a tryptic peptide of the α2NC1 domain of mouse collagen IV [α2(IV)NC1]. Shown are MS² spectra of unmodified (A) and brominated (B) peptide from the α2NC1 domain, including peak assignments. The mass shifts (ΔM) in y-ion series starting from y⁵ and indicating the sequence position of brominated tyrosine residue are also shown. Masses of ⁷⁹Br and ⁸¹Br isotopes calculated from the data in A and B were 78.9107 ± 0.0271 and 80.9086 ± 0.0143, respectively. The standard isotopic masses for ⁷⁹Br and ⁸¹Br are 78.9183 and 80.9163, respectively. (Insets) The sequence of unmodified (A) and brominated (B) precursor peptide and the MS² fragmentation pattern producing specific y- and b-ions. The y axis in A and B is relative abundance. (C) Quantitation of the same brominated peptide in α2(IV)NC1 preparations from WT and Pxdn^−/− mice. Normalized relative abundance represents modified peptide/(modified peptide + unmodified peptide). The WT data represent mean ± SD for three independent kidney NC1 preparations; the Pxdn^−/− mouse data are from a preparation generated from the pooled kidneys of five Pxdn^−/− mice.