Efficient enrichment and identification of phosphopeptides by cerium oxide using on-plate matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

An efficient and simple method for enrichment and identification of phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using cerium oxide is presented. After pretreatment of tryptic digests of phosphoproteins with CeO(2), nonphosphopeptides are discarded and phosphopeptides are enriched. By applying the separated CeO(2) on a target plate and analysis using MALDI-TOF MS, peaks of phosphopeptides and their correspondingly series of dephosphorylated peptides are observed in the mass spectra. Thus, the phosphopeptides are very easy to identify with the mass difference, which are all 80 Da between adjacent peaks in the same series, and clear background in the spectra owing to elimination of signal suppression from large amounts of nonphosphopeptides. Furthermore, the phosphopeptides can be dephosphorylated completely after a further NH(4)OH elution. Tryptic digest products from several standard proteins are pretreated using CeO(2) to demonstrate the efficiency of this method. Phosphopeptides from a very small quantity of human serum are enriched and analyzed, and proteins also identified by searching against a database using Mascot on MALDI-TOF/TOF fragments, which indicates that this method may be employed in complex samples for further application.