Analysis of proteomics

Peptide hits in each gel slice (Supplemental Tables S3 and S4) were compared with the Saccharomyces cerevisiae protein database using the SEQUEST algorithm (performed by Midwest Bio Services) and curated with the Protein Information Resource Peptide Matching analysis tool (http://research.bioinformatics.udel.edu/peptidematch/index.jsp). The data from individual slices were then combined and sorted to generate a composite list of hits for wild-type or the auxilin-depletion vesicle preparation (Supplemental Tables S5 and S6). Finally, to generate a list of protein hits specific to the auxilin-depletion strain, all of the gene products identified in the wild-type strain were subtracted from the hits obtained from the auxilin-depletion strain (Supplemental Table S7). During the purification of coated vesicles, RNase was added to the cell extract to remove polysomes (Lemmon et al., 1988 ), but ribosomal subunits and translation factors were still a common contaminant in the Sephacryl S-1000 coated-vesicle column fractions. Therefore all proteins involved in translation, including translation factors and ribosomal subunits, were excluded from the auxilin-depletion strain list.