Pulse labeling of selected glycoproteins. (A–E) Blue and black segments designate signal sequences and TM spans respectively; red lines are disulfides. The disulfide pattern for KDEL2 is unknown. Color-coded symbols are defined below panels A–C. EH designates digestion with EH. Asterisks in panels C and E indicate sites that were quantified in a single LC-MS/MS experiment. Pulse labeled glycoproteins (10 min, 35S Trans label) were immunoprecipitated, resolved by SDS-PAGE, and quantified by phosphor image analysis (see Materials and methods). (A–C and E) Quantification is for the experiment shown, which is representative of two or more pulse labeling experiments. (D) The MEGF9 5NQ mutant lacks the acceptor sites outside the EGF_3 repeats (green blocks). As indicated, cells were preincubated with 3 mM DTT for 5 min before and during the pulse labeling period. The vertical line designates images derived from separate experiments aligned to show comparable mobilities for glycosylated MEGF9 and EH-digested MEGF9.
