Fig. 1.

Phosphoproteome analysis by 4-step elution from a fully automated phosphopeptide purification system. A sample protein denatured with 8 M urea was diluted 4 times to reduce the urea concentration and then digested with trypsin. After acidification and removal of insoluble peptide by centrifugation, the thus prepared peptide mixture was directly applied on a fully automated phosphopeptide purification system and eluted in 4-steps. After drying and desalting, the thus purified phosphopeptides were analysed by LC MS/MS.