Abstract: It is fundamental to understand fragmentation property and behavior of large peptides under different fragmentation conditions. More importantly, it is essential and practical in study endogenous biological peptides to know the experimental condition and limitation of different mass spectrometry fragmentation techniques in analysis of large peptides. We conducted a systematical investigation of large peptide fragmentation using a chemically synthesized peptide ladders with several commonly used fragmentation techniques, including resonant excitation low energy collision induced dissociation, electron transfer dissociation, and high energy collision induced dissociation. Peptide fragmentation efficiency and pattern were tested against amino acid composition, peptide length, and charge stage, as well as different ionization methods, e.g. electrospray ionization and matrix assistant laser desorption ionization. Detailed experimental findings will be reported.