High-throughput protein identification in most proteomic researches relies primarily on mass spectrometry (MS) analysis of the large-scale digested peptides, which has routinely been conducted in the positive ion mode on protonated peptides. Nevertheless, there is still a subset of proteins or peptides rich in acidic amino acids which are underrepresented in current popular strategy. Negative-ion MS has provided a potential to achieve a full proteome coverage largely due to the complementarity to its positive-ion partner. However, to date the available knowledge of the fragmentation behaviors of deprotonated peptides is insufficient towards to an automatic interpretation as that done for protonated peptides in bottom-up proteomics. Here, we statistically characterized the fragmentation patterns of 36 synthesized peptides, containing 4-16 amino acids, in the negative ion mode using CID and HCD under a variety of MS conditions, including a series of normalized collision energies (NCE). In light of the statistical analyses on all MS/MS spectra acquired in our study, the relationships between peptide fragmentation efficiency and its NCE were depicted for the first time. To facilitate communication, we modified the nomenclature of fragment ions in the negative ion mode in reference to that in the positive ion mode. Our studies find that y-, c- and z-type ions are the most dominant species in both CID and HCD spectra of deprotonated peptides, accompanied by abundant neutral loss peaks. Furthermore, HCD is generally better than CID for peptide sequencing in the negative ion mode, since HCD generates more backbone cleavage products whereas CID produces more side-chain neutral loss peaks. For disulfide-bonded peptides and C-terminally amidated peptides, specific fragmentation patterns are observed and analyzed. These statistically significant fragmentation behaviors of deprotonated peptides reported in our study will promote further investigation of the fundamental mechanisms and facilitate algorithmic development for peptide sequencing in the negative mode.