Phosphorylation of a specific site is a key factor for the activity of many proteins. For the determination of the presence and position of a phospho group on a peptide, mass spectrometry (MS) is a valuable tool. Modifications can be detected by Edman degradation via derivatization or radioactive labelling of the phospho group. Without MS detection, the location of phospho groups is difficult at best, if indeed possible.

In linear MALDI an indication of a phosphorylated peptide in a digest mixture is given by the difference in intensity of the peaks obtained in the positive and the negative ion mode. However, other peptides carrying highly negatively charged groups usually also result in peaks with higher intensity in the negative than in the positive ion mode. More specific methods are needed to distinquish phosphopeptides from the other peptides.

We report a method using MALDI metastable decay to identify the presence of phospho peptides in a digest mixture. The ions from a peak of interest - as indicated in the linear spectrum - are isolated by fast pulses and only these ions are allowed to enter the reflector. The resulting metastable spectrum is inspected for a peak at ca. 97 Da below the molecular ion peak. This peak is diagnostic of phosphopeptides and originates from the loss of the phospho group from the molecular ion between the acceleration region and the entrance of the reflector.

Subsequent analyses performed in the positive ion mode using the MALDI metastable decay method yields sequence ions from fragmentation of the peptide backbone. Correlating the masses of the metastable ions spectrum with the calculated ones from the known sequence the phosphorylation site can be assigned to one specific amino acid residue in the sequence. MALDI metastable decay is a practical, efficient method for location of phosphorylation sites in peptides and proteins.