Validation of O-GlcNAcylation site mapping. (A, C, and E)FETDMS/MS spectra recorded on (A) [M + 4H]+4 ions (m/z 815.6) for the modified peptide, gSTEANVLPPSgSIGFTFSVPVAK, from NUP153; (C) [M + 3H]+3 ions (m/z 531.9) for the modified peptide, TITVPVgSGSPK, from EMSY; (E) [M + 3H]+3 ions (m/z 499.2) for the modified peptide, SAPAgSQASLR, from NUMA. Abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val. Predicted m/z values for product ions of type c′ and type z′•(monoisotopic and average masses for singly and doubly charged ions, respectively) are listed above and below the peptide sequence. Observed product ions are underlined and also labeled in the spectrum. Ions in the precursor isolation window are labeled with a triangle (). Brackets enclose ions corresponding to charge-reduced species, as well as fragments derived from these charge-reduced species. Product ions resulting from loss of an aminomethyl triazole radical are labeled with a circle (●). Insert displays the isotopic distribution, calculated monoisotopic mass, and experimental monoisotopic mass of precursor ions corresponding to the labeled and unlabeled forms of the indicated peptide. (B, D, and F) Nup153, NuMA1, or EMSY were immunopurified from lysates of thymidine-nocodazole synchronized cells overexpressing either GFP or OGT. Precipitates were washed, separated on SDS-PAGE, and transferred to membranes for blotting. Membranes were probed for O-GlcNAc. For Nup153 and NuMA1, the increase in O-GlcNAc in the samples from the OGT-overexpressing cells is evident. For EMSY, the increase is not readily apparent, which may be due to only one of seven O-GlcNAcylation sites exhibiting increased modification.