Two alternative proteinases, Asp-N Sequencing Grade and Glu-C Sequencing Grade are available from Promega to expand options for protein digestion. Asp-N, Sequencing Grade, is a metallo proteinases that preferentially cleaves at the N-terminus of aspartic and cysteic acid residues. Glu-C is a serine proteinases that cleaves with high specificity at the C-terminus of glutamic and aspartic acids. Use of alternative enzymes for protein digestion increases the confidence in mass spectrometry data by confirming the protein sequence and aids in the mapping of post-translational modifications (PTMs). Here we describe the cleavage specificity of endoproteinases Asp-N and Glu-C, and show an example of improved protein sequence coverage through the use of alternative digestion with these enzymes.