Abstract: Alginate homooligosaccharides including oligomannuronate (M) and oligoguluronate (G) with degree of polymerization from 2 to 7 were analyzed by linear ion trap-Orbitrap mass spectrometer (LTQ Orbitrap XL) using collision induced dissociation (CID) and high energy collision induced dissociation (HCD). First of all, comparison of CID MS/MS and HCD MS/MS of either M or G was conducted. Taking the trimannuronate (M3) as an example, CID MS/MS showed the m/z 369, 193 and 175 ions were assigned to C₂/Y₂, C₁/Y₁ and B₁/Z₁, respectively, which were formed due to single glycosidic bond cleavage, while HCD MS/MS caused cleavage of all glycosidic bonds and showed weak A-type ions, such as ^0,2A (m/z 309, 485), ^2,5A (m/z 291, 467) and ^0,4A₃ ions (m/z 471). Interestingly, an internal M produced a Z_int-CO₂ ions at m/z 307, which was unique and found only in HCD MS/MS. Furthermore, CID MS/MS and HCD MS/MS of M3 and triguluronate (G3) were executed. For CID, M3 and G3 did not show significant difference, and they gave identical glycosidic bond cleavage ions C₁ (m/z193), B₁ (m/z 175) together with the dehydrated and decarboxylated ions (m/z 157 and m/z 131). These results suggest that it is impossible to differentiate the two residues at a nonreducing terminal of M3 and G3 by CID. However, a significant difference was observed in HCD spectra for M3 and G3. It was found that besides decarboxylation fragment m/z 307, debris ^2,4A₃ (m/z 411), ^2,5A₃ (m/z 467) and ^0,4A₃ (m/z 471) were found in M3, whereas these ions were not found in HCD MS/MS of G3, thus demonstrating that HCD MS/MS can be used to differentiate M3 and G3. In the CID MS/MS of pentamannuronate (M5), fragment ions ^0,2A₃ (m/z 837), ^0,4A₃ (m/z 823) and ^2,5A₃ (m/z 819) was found apparently, however, HCD displayed rich fragment ions in the low mass region, which should be ascribed to the characteristics of high-energy dissociation of HCD. For heptamannuronate (M7) and heptaguluronate (G7), doubly charged protonated species were collected as parent ions for CID and HCD. HCD MS/MS provided more fragment ions than CID MS/MS, especially, Z_int-CO₂ ion (m/z 307, 483, 659, 835 and 1 011) signal was apparently showed in M7 HCD spectra. Taken together, this work demonstrates that HCD MS/MS provides rich product ions and has no low mass cut off. The fragment ions in HCD spectra have high mass accuracy and resolution. These characteristics of HCD spectra complement the power of CID and allow easy spectrametric interpretation and high confidence in structural elucidation for alginate oligosaccharides.