Figure 2

Identification by mass spectrometry (MS) of in vivo O -GlcNAc sites in K-RTA. (A) Affinity purification of FLAG-K-RTA by M2-FLAG resin. Protein extracts from 1 g (~10⁸ cells) of Dox-treated 293TetKR cells were used as starting material. Purification was verified by western blot analysis using M2-FLAG antibody. ST, starting material; FT, flow through; W, wash; E1-E4, eluted fractions 1-4. (B) Purified K-RTA and K-RTA mutant defective in nuclear localization signal (NLSm) were readily immunoreactive to the anti-O-GlcNAc antibodies RL2 and CTD110.6. Aliquots of ~ 50 ng of each affinity-purified protein, including K-RTA, K-RTA NLSm (NLSm), EBV Rta (E-RTA), rhesus monkey rhadinovirus RTA (R-RTA) and 300 ng bovine serum albumin (BSA), were used to prepare one of the three identical immunoblots. The blots were respectively hybridized to anti-FLAG, RL2 and CTD110.6. Whereas the four FLAG-tagged proteins were obviously detected by anti-FLAG, only K-RTA and NLSm possess O-GlcNAc epitopes that were detectable by RL2 and CTD110.6. (C) MS/MS spectrum of a candidate singly O-GlcNAcylated tryptic peptide, AVAQGAVITATTVPQAMPAR, from the E2 fraction of the purified K-RTA (rectangle) at m/z 724.70 (3+). The strong oxonium ions at m/z 204 (HexNAc*), 168 and 138 confirm the presence of GlcNAc moiety but the precise site location cannot be established unambiguously because of the facile neutral loss of O-GlcNAc (see text).