A rapid, picomole-scale method is described to locate phosphorylation sites in phosphoproteins by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with enzymatic modification of the analyte. There are three steps to locate phosphorylation sites in a phosphoprotein: (i) degradation of the phosphoprotein into small peptides by specific enzymatic or chemical reactions; (ii) identification of the phosphopeptides by (-\mathbf{8 0}) (or multiples of -80)-Da mass shifts in the mass spectra after dephosphorylation with alkaline phosphatase; (iii) location of the phosphorylation sites by mass mapping. As the size of the protein increases, it is advantageous to fractionate the mixture by HPLC and analyze each fraction by MALDI-TOF-MS. To perform mass mapping, the primary structure of the protein must be known. Bovine (\beta)-casein was analyzed by this method. The conclusions about the specific phosphorylation sites of bovine (\beta)-casein from our data coincide with previously reported results. From calculations, it is found that a mass spectrometer with (0.1 %) mass accuracy is sufficient, for mass mapping, to identify completely or partially digested tryptic peptides in the mass range of 100 (\mathbf{8 0 0 0}) Da from bovine (\beta)-casein (MW 23,983 ). C 1994 Academic Press, Inc.