Comprehensive identification of novel post‐translational modifications in cellular peroxiredoxin 6

Peroxiredoxin 6 ( PRDX 6), a 1‐ C ys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A 2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post‐translational modifications ( PTM s) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTM s in PRDX 6 utilizing proteomic tools including nano UPLC‐ESI ‐q‐ TOF MS/MS employing selectively excluded mass screening analysis ( SEMSA ) in conjunction with MOD i and MOD map algorithm. We chose PRDX 6 obtained from liver tissues from two inbred mouse strains, C 57 BL /6 J and C 3 H/H e J , which vary in their susceptibility to high‐fat diet‐induced obesity and atherosclerosis, and a B 16 F 10 melanoma cell line for this study. When PRDX 6 protein samples were separated on 2D‐PAGE based on p I , several PRDX 6 spots appeared. They were purified and the low abundant PTM s in each PRDX 6 spot were analyzed. Unexpected mass shifts (Δ m = −34, +25, +64, +87, +103, +134, +150, +284 D a) observed at active site cysteine residue ( C ys47) were quantified using precursor ion intensities. Mass differences of −34, +25, and +64 D a are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and C ys‐ SO 2 ‐ SH , respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 D a) as well as unknown modifications (+134, +150, +284 D a). Comprehensive analysis of these PTM s revealed that the PRDX 6 exists as a heterogeneous mixture of molecules containing a multitude of PTM s. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX 6 from mouse liver tissues included, among others, mono‐ and dioxidation at T rp and M et, acetylation at L ys, and deamidation at A sn and G ln. Comprehensive identification of the diverse PTM s occurring in this bifunctional PRDX 6 enzyme should help understand how PRDX 6 plays key roles in oxidative stresses.