PMF consists of three steps. (1) The preparation of the biological sample: a band or a spot of the electrophoretic gel is selected and digested by a suitable protease, such as trypsin. The resulting mixture of peptides is analyzed with a mass spectrometer, usually in MALDI-TOF configuration. (2) A reference protein database is created, reproducing in silico on a set of known proteins the step 1, considering also possible missed cleavages and post-translational modifications. (3) The acquired spectrum is matched against the theoretical spectra generated by in silico digestion of all proteins in the reference database (step 2) and a ranked list of candidate proteins is obtained.