Phosphopeptide shotgun analysis using mass spectrometry and Phos-tag agarose beads

Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.