Mannosidic glycans on misfolded Arabidopsis SUBEX glycopeptides

The mutated extracellular domain of Arabidopsis STRUBBELIG (SUBEX) was fused to GFP and either transiently expressed in Nicotiana benthamiana leaves or stably in Arabidopsis thaliana seedlings and purified. The purified SUBEX proteins were subjected to SDS-PAGE and CBB staining. Corresponding gel pieces were excised and the proteins digested with proteases and jack bean alpha-mannosidase. Glycopeptides were analysed by MS to identify the N-glycan structures. In addition, glycans were released from SUBEX peptides using PNGase A and analysed by PGC-LC-MS.