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Infrared Multiphoton Dissociation (IRMPD)

Infrared multiphoton dissociation (IRMPD) of [M+Li]+ and [M+Na]+ ions from glycans implemented in ICR instruments appears to give very similar fragmentation to that observed by CID [272] and enables fragmentation of larger N-linked glycans to be observed when it could not be obtained by CID [273]. IRMPD of [M+nH]”+ ions of large glycopeptides, on the other hand, show preferential fragmentation of the peptide chain [274]. Positive ion IRMPD spectra of these O-linked alditols, as with the N-glycans, are very similar to low-energy CID spectra [275].


Sequential fragmentation or “disassembly” has also been used to obtain structural information on N-glycans. Several successive stages of fragmentation can be observed in an ion trap instrument, particularly where cleavages can occur adjacent to GlcNAc residues [144, 276]. The technique is particularly valuable when interfaced with HPLC [175]. MS” spectra have shown that many of the fragment ions in the MS2 spectra from complex glycans arise from several pathways, a property that can present problems for the interpretation of “unknown” spectra [262].

Ashline et al. [277] have studied the fragmentation of small permethylated oligosaccharides as alkali metal adducts and identified ions that are characteristic of, for example, the nature of the terminal monosaccharide residue. They [278] have also used the technique with fragmentation stages up to MS7 to investigate the occurrence of isomers from N-glycans released from glycoproteins such as chicken ovalbumin and IgG. 2-AP-labeled N-linked glycans have been studied by Ojima et al. [279] using a MALDI-QIT-TOF instrument. The work suggested that some isomer information is available in the form of peak intensity in the positive ion MS3 spectra. Information on anomeric configuration has been obtained with energy-resolved spectra at various stages of MS"

[280]. It was noted that for the [M+Na]+ ions from linear oligosaccharides, a- linkages fragment at a lower energy than p-ones. However, the authors were not sure if the relationship would apply to all types of carbohydrate.

MS" experiments have provided information on both the glycan and peptide moieties of glycopeptides. Thus, for example, Deguchi et al. [281] have chosen both peptide- and carbohydrate-derived ions from the MS2 spectra of these compounds and fragmented each at the MS3 stage in both positive and negative ion modes to obtain data on both halves of the molecule. In another approach for the analysis of glycoproteins, the compounds were first digested in-gel with trypsin followed by MS" experiments in an ion trap-TOF instrument. Information on the carbohydrate was obtained in the early stages of fragmentation until finally all of the sugar had been removed, allowing the peptide to be sequenced by a series of y ions [282].

Although acquisition of MS" spectra is usually performed with trapping instruments, it is possible to use in-source fragmentation to provide the MS2 spectrum and then to fragment the resulting fragments in a collision cell in the conventional manner to provide the MS3 spectrum [283]. However, the method is only satisfactory for single compounds when there is no ambiguity as to the source of the MS2 fragments.

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