SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.