Abstract: To comprehensively compare the difference of collision induced dissociation (CID) and high energy collision dissociation (HCD), the tryptic digests of bovine serum albumin (BSA) and cell lysates were analyzed by LTQ-Velos Orbitrap. The HCD fragmentation identified more peptides and this mode showed higher MS/MS success rate and higher Mascot score in the tryptic digests of BSA. The results demonstrated that the HCD fragmentation has higher MS/MS spectra quality than CID. But in the tryptic digests of cell lysates, the CID fragmentation identified more MS/MS spectra, peptides and proteins, which indicated that it produces higher sensitivity. The HCD fragmentation shows higher MS/MS success rate and higher Mascot score distribution, demonstrating higher quality of MS/MS spectrum. Therefore, based on these findings, both CID and HCD modes could be used for large-scale proteomic analysis. The CID mode has higher sensitivity, and HCD mode can achieve higher quality of MS/MS spectrum.