Distinguishing of Linkage Isomers of Lactotetra Oligosaccharides by Using the Relative Ion Intensity Analysis of Post-source Decay

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to analyses of biomolecules, such as proteins, peptides and oligosaccharides.1–5 The post-source decay (PSD) fragmentation of peptides and oligosaccharides occurred at peptide bonds and at glycosyl linkages in MALDI-MS, respectively. Thus, the PSD mass spectra are useful for the sequencing of peptides and saccharides. The sequencing of peptides could be determined by this method because the peptide bond is just one type, and because almost all amino acids have different molecular weights. On the other hand, it is very difficult to determine the fine structure of the saccharide chains by MS because of the existence of many structural isomers with different linkage types. We could obtain and discuss only the simplest sequence information of oligosaccharides, like Hex-HexNAc-Hex-Hex. In the MALDI-PSD fragment mass spectra of some isomers of oligosaccharides, different tendencies in the spectral patterns were observed;6–8 however, it has been very difficult to quantitatively discuss the ion intensity by using a conventional TOFMS.8 Unfortunately, it is impossible to compare the ion intensities in the total PSD spectra because these spectra are usually acquired in segments by stepping the reflectron voltage. Sequence information can be extracted from the reconstructed time-of-flight mass spectrum by scanning the voltage of the ion reflector and bringing narrow segments of the product spectrum into focus. Consequently, the signal strength might differ in different segments where the ionization conditions might differ. Although it might be possible to compare the absolute intensities of PSD ions in only a narrow segment, huge accumulations of the actual measurements were required to obtain high reproducibility in the absolute intensity; also, the actual spectra of the huge measurements were hardly measured under the same conditions. A logical and actual solution to this problem would be to use an instrument equipped with a reflectron, which records all fragment ions in a single spectrum. A new type of reflector of curved-field reflectron in a TOF mass detector enables the simultaneous focusing of a wide mass range of metastable fragment ions and the observation of the entire PSD fragment spectrum in a single experiment.9,10 Thus, the product-ion mass spectra can be collected without an adjustment under the same conditions, thereby eliminating the need to scan the reflector voltage. Therefore, the relative intensity of the PSD fragment ions can be discussed at an accurate level. In this work, we studied a new method of a structure analysis for the glycosylation and/or glycoconjugates using the ion intensities of the PSD fragment ions in the curvedfield reflectron MALDI-TOF mass spectra. Previously, we studied the PSD fragment spectra of sugarbranched cyclodextrin derivatives by using curved-field reflectron MALDI-TOFMS.11–15 The PSD ions produced from the branch had higher intensities than those from the part of the cyclodextrin, which is cyclo-malto-oligosacchraide. The former 83 ANALYTICAL SCIENCES JANUARY 2001, VOL. 17 2001 © The Japan Society for Analytical Chemistry