Fig. 4. Elucidation of the C-terminus of NTF-NgR by mass spectrometry. (A) SH-SY5Y cells expressing NgR were incubated for 48 hours in OptiMEM. NTF-NgR was immunoprecipitated from medium using the anti-V5 antibody, subjected to SDS-PAGE and visualised by staining with Coomassie Blue. The band corresponding to NTF-NgR (*) was excised from the gel along with a control blank gel slice, digested in-gel with Lys-C and the resulting fragments analysed by MALDI-TOF mass spectrometry. Bands corresponding to the heavy (H) and light (L) chains of the anti-V5 antibody in the immunoprecipitate are indicated. (B) Table showing the predicted mass and sequence of peptides generated by Lys-C digestion of human NgR and their detection by MALDI-TOF mass spectrometry. Amino acid residues correspond to those for un-tagged human NgR. The mass of the peptide corresponding to residues 423-447 could not be predicted due to the presence of the GPI anchor. The peptide of mass 26980.21 was too large to be extracted from the gel. (C) Detail of the MALDI-TOF mass spectrometry spectra showing the peak of mass 1396.72 present in the NTF-NgR digest (lower spectrum) but not the control sample (upper spectrum) predicted to correspond to the indicated sequence. (D) Table showing the mass and predicted sequence of subunits detected following MS/MS analysis of peak 1396.72. Amino acid residues correspond to those for un-tagged human NgR.