 Solution

Analyze the corresponding region from a parallel preparative SDS-PAGE gel, digest the recovered proteins and identify peptides by liquid chromatography-mass spectrometry. The lane and gel region should match the $35\mathrm{kDa}$ signal, with an adjacent-background or control-sample region processed in parallel. Because several proteins can comigrate, validate the candidate against the actual phospho-band, for example by immunoprecipitating the candidate and testing Ras-PST25 reactivity, using a target-specific Western blot, or showing disappearance of the band after specific depletion. Phosphopeptide identification provides additional evidence; mass alone or detecting a common contaminant in the excised region is insufficient.