When very broad coverage of a protein is required, for example in post-translational modification (PTM) mapping, the use of a combination of two or more proteases could be very useful. Often some regions of the protein are not accessible to a single protease. Two alternatives to trypsin are chymotrypsin and endoproteinase Glu-C (protease V8 from Staphylococcus aureus). This protocol describes the use of DigesTip Trypsin, DigesTip Chymotrypsin and DigesTip Glu-C in combination: the use of at least two different DigesTip, chosen from those above, increases significantly the number of peptides obtained, leading to a higher protein sequence coverage.