FIG. 2.

Identification of DHA in H₂O₂-treated GPx1 by MS. (A) Purified human GPx1 was subjected to SDS-PAGE analysis on a 14% gel. Size markers are indicated. (B) Purified Gpx1 (30 μg) was incubated in the absence or presence of 1 mM H₂O₂ for 1 h at 37°C and then assayed for GPx1 activity. Data are means ± SD of triplicates from a representative experiment. (C) GPx1 incubated with (dark gray line) or without (light gray line) H₂O₂ as in B was subjected to tryptic digestion, and the resulting peptides were fractionated by HPLC on a C₁₈ column. Peaks eluting between 39 and 40 min are shown. (D) Expanded LC-ESI-Q-TOF tandem MS spectra for peptides corresponding to peak 1 (left panel) or peak 2 (right panel) from C. The isotopic distribution was normalized relative to the largest peak. (E) Tandem MS spectrum obtained from fragmentation of the doubly charged ion with an m/z of 827.4 from peak 2 in D. The y ion series defined the indicated amino acid sequence. The mass difference of 69.0 Da between the y5 and y6 ions defined residue X as DHA.