Alkaline Phosphatase Treatment of Phosphopeptides: In-Solution Dephosphorylation prior to MALDI-MS Analysis

INTRODUCTIONThe use of the enzyme alkaline phosphatase allows identification of phosphopeptides in a mixture of predominantly nonphosphopeptides. Using a MALDI-MS instrument, the masses of peptides are acquired both before and after alkaline phosphatase treatment, which removes phospho-moieties from serine, threonine, and/or tyrosine. (Any peptide whose mass decreases by 80 Da, or a multiple thereof, is a phosphopeptide.) An advantage of using MALDI-MS for these experiments is that the peptide ions produced tend to be singly charged rather than multiply charged (as with ESI), thus making the interpretation easier. This protocol describes a method for in-solution dephosphorylation prior to MALDI-MS analysis.