Formalin-Fixed Paraffin-Embedded (FFPE) samples are treasure for proteomic studies of disease. However, their usage is hampered for proteomics study because of the crosslink among proteins, and protein vs nucleic acid. Even worse, other covalent chemical modifications like methylation introduced by formaldehyde interfere trypsin digestion. We applied LysargiNase for the first time in FFPE samples, and systematically compared the samples digested by LysargiNase with commonly used tryptic samples. A total of 33095 peptides and 3559 proteins were identified with LysargiNase and trypsin combined in two replicates. LysargiNase increased peptide identification by 19.3% and protein identification by 13.3% on the basis of trypsin. Consistently, LysargiNase increased C terminal peptide identification by 47.7%. Moreover, LysargiNase showed less missed-cleavage rate (54.5%) at methylated sites than trypsin (74.5%), contributing to the identification of methylated peptides in FFPE samples.