An SDS-PAGE gel band containing ~5 μg of RET protein was excised and washed with 50% acetonitrile/100 mM ammonium bicarbonate. The protein sample was reduced with 10 mM dithiothreitol/100 mM ammonium bicarbonate and then alkylated using 15 mM iodacetamide/100 mM ammonium bicarbonate. After washing twice with 40% acetonitrile/200 mM ammonium bicarbonate, the RET gel band was deglycosylated with peptide N-glycosidase F. Trypsin and chymotrypsin were added for protein fragmentation, which were extracted with 0.1% trifluoroacetic acid and 60% acetonitrile. The resultant digests were loaded onto the Dionex U3000 NanoLC for ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) analysis using the Qstar Elite mass spectrometer. The mass spectrometry data were searched against the RET sequence using the Mascot search engine. Asparagine residues predicted to be glycosylated were changed to aspartic acids in the search sequence. Eleven of 12 predicted glycosylation sites in the RETECD were glycosylated in this sample: Asn98, Asn151, Asn199, Asn336, Asn343, Asn361, Asn367, Asn377, Asn394, Asn448, and Asn468. No glycosylation was detected at Asn554. Peptide mapping for cross-linked GFRα2, RET, and NRTN was carried out in a similar fashion, except that a Sciex X500B mass spectrometer was used.

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