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Reputable Mentor II
Reputable Mentor II
Davea KA, Hamilton BR, Wallis TP, Furness SGB, Whitelaw ML, Gorman JJ.
Int. J Mass Spectrom. 2007Jun;268:168-180.
Analysis of a tryptic digest of a recombinant D83A mutant of the murine dioxin receptor (D83A-DR) by CapHPLC-MALDI-TOF/TOF-MS/MS revealed a peptide corresponding to cleavage at a lysine that had either been mutated to an arginine or modified by dimethylation or formylation (Ser-Phe-Phe-Ala-Val-Ala-Leu-Me2K/FormK/Arg). High mass accuracy data were obtained for the pseudomolecular ions of synthetic peptides corresponding to the potential alternate structures of the variant D83A tryptic peptide and their CID products using an LTQ-Orbitrap-FTMS instrument. The high mass accuracies obtained by both FT-MS and FT-MS/MS were more than sufficient to enable differentiation between the synthetic peptides. The only potential fragmentation characteristic produced by CID was the formation of ions at −17 u relative to y ions of the Arg peptide. The variant D83A-DR tryptic peptide was also detected by CapHPLC-ESI-LTQ-Orbitrap-FT-MS and MS/MS with masses detected for the pseudomolecular and CID product ions being consistent with dimethylation of a C-terminal lysine. Fragmentation of the synthetic peptides by MALDI-TOF/TOF-MS/MS produced a range of fragmentation characteristics which provided a basis for distinguishing between peptides with C-terminal Me2K, FormK and Arg. These characteristics included specific immonium ions, apparent side chain fragmentation from the precursors of 28 or 30 u and satellite ions at −17 u relative to the y ions of the Arg peptide. Comparison of the fragmentation properties of the D83A-DR derived tryptic peptide produced by MALDI-TOF/TOF-MS/MS with those of the synthetic peptides corroborated the mass accuracy-based assignment of a dimethylated lysine at the C-terminus of the D83A-DR tryptic peptide. This represents the first documentation of any post-translational modification of DR, other than two previously described sites of phosphorylation.
Protein Discovery Centre, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Queensland 4029, Australia
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