Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional...

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.