Pre-coated analysis substrates, and methods of making the substrates and using them to analyze animal tissue, are described. The pre-coated analysis substrates can be made by forming a matrix surface on an analysis substrate; adding a protease to the matrix surface to form a pre-coated analysis substrate; and placing an animal tissue specimen on the matrix surface. The animal tissue can then be analyzed by allowing the protease to partially digest the animal tissue specimen; and analyzing the partially digested animal tissue specimen by mass spectrometry.