Fig. 1.

Schematic illustration of workflow. Skeletal muscle is dissected (1) and subjected to subcellular fractionation to enrich for membrane proteins (2). Glycoproteins and associated proteins are isolated using wheat-germ agglutinin (WGA) chromatography (3), and are separated by sucrose-gradient fractionation (4). Fractions of interest are spiked with cysteine carboxymethylated tryptic BSA peptides and subjected to ion-exchange chromatography to reduce sample volume; they are eluted using increasing salt concentrations (5). A first LC/MS run is performed on each elution fraction (6), and the peptides identified are used to generate a directed list (7). A directed LC-MS/MS run is then performed to provide quantitative data (8). Proteins are annotated based on queries of the Spectrum Mill database (9). The annotated quantitative data is then curated (10).