Preliminary Studies On Derivatived Oligosaccharide Molecular Ions Fragmentation Pattern In ESI-MS~n Analysis

Biomass spectrum technology has been widely used in the analysis of carbohydrates during the past two decades, and has become a powerful tool for qualitation, quantitation, and structure investigation of glycans. Studies on glycan fragmentation pattern and law is a significant work for carbohydrate structure elucidation, which is based on collision-induced glycan fragmentation in mass spectrometry. To increase and develope the knowlege about regular fragmentation pattern of glycans and provide essential reference data for their structure analysis, fragments of several oligosaccharide derivatives of different structures and connections in electrospray mass spectrometry were investigated in detail, and the results and conclusions are as follows:1. Oligosaccharides permethylated and analyzed by ESI-MSnCiucanu and Kerek's permethylation method were employed here to study seven different oligosaccharides, the permethylated oligosaccharides were analyzed by ESI-MSn, and studied the reproducible ESI-MSn results to figure out the law of collision-induced fragmentation.1) ESI-MSn analysis of permethylated glycans with different composition and in the same linkageLactose Gal-β(1-4)-Glc and Maltobiose Glc-α(1-4)-Glc were in the same linkage but with different composition were produced the same fragments by ESI-MS2, m/z 445, m/z415, m/z 329, m/z 259 and m/z 241, in which m/z 445 and m/z241 produced by lactose is twice higher than that of maltobiose. In ESI-MS3, In ESI-MS3, Only Maltobiose produced two different crossing ring cleavage fragments, m/z 315 (2'4A2) and m/z 372 (0,4A2), but lactose did not. The relative abundance of maltobiose m/z 259 was 90, while lactose was only one-third of the former. 2) ESI-MS" analysis of permethylated glycans with different linkage and in the same compositionSignificant differences were also found between permethylated sucrose [Glc-α(1-2)-Fru] and turanose [Glc-α(1-3)-Fru] in MS2 spectrogram, which have the same composition but different linkage. B1 or C1 cleavage of sucrose [Glc-α(1-2)-Fru] occurred, and produced the peaks of m/z 259 and m/z 241, while turanose [Glc-α(1-3)-Fru] generated m/z 245 and a higher peak of m/z 431. But both saccharides generated m/z 359.In MS2 analysis between permethylated melibiose[Gal-α(1-6)-Glc] and lactose[Gal-β(1-4)-Glc], melibiose generated two sets of crossing ring peak(m/z 301 and m/z 345, m/z 329 and m/z 373) which mass difference was 44Da, and lactose produced two crossing ring peak m/z 329 and m/z 372. In addition, lactose occurred B1 or C1 cleavage which only produced crossing ring peak m/z 241, while melibiose was not. So, the linkage of glycans were made a significant difference in MS2 analysis.Several disaccharides used in this study were isomers, such as maltobiose, lactose, melibiose, turanose and sucrose. These disaccharides were permethylated and subjected to MSn analysis reproducibly could be used for the identification of isomers.3) MSn analysis of trisaccharide and tetrasccharide which contain structures like the bisaccharide above.Permethylated raffinose[Gal-α(1-6)-Glc-α(1-2)-Fru], sucrose[Glc-α(1-2)-Fru], stachyose[Gal-α(1-6)-Gal-α(1-6)-Glc-α(1-2)-Fru], turanose[Glc-α(1-3)-Fru] all produced m/z 359 and the relative abundance of m/z 359 was 100 in MS3 analysis of raffinose and MS4 analysis of stachyose, but the others were significantly different, indicating that both raffinose and stachyose were containing fructose.Comparing the difference between MS3 of raffinose and MS4 of stachyose, all peaks they produced were identical except m/z 375 produced in the former one. These results of MS data can be used to infer the structure of glycans.2. ESI-MSn analysis of UV and fluorescent reagents derivatived oligosaccharide Carbohydrates did not have chromophore, in order to facilitate HPLC and LC-MS analysis, it is necessary to derivatize glycans with amino-aromatic reagent to make it bring an UV or fluorescence groups, and these glycan derivatives can be more sensitively detected by mass spectrometry. Eight most commonly used UV and fluorescent derivatizing reagents were employed to analyze disaccharide isomers, derivatized glycan samples of high purity were obtained, and a good signal-to-noise of 6-10pmol in the ESI-MS" mass spectrometry experiments was got.1) The study found that disaccharide labeled by a variety of reagents mainly produced the Y and Z ions in MS" analysis, and B and C ions did not frequently occured. In the multistage mass spectrometry of disaccharide labeled by various reagents, L and M labeled by the same reagents were obviously different, which indicated that the mass spectrum could be identified according to two kinds of disaccharide isomers. We also discovered that pyranosylene ring formed by either glycosidic bond cleavage or dehydration tends to break across the ring, and the cracking reaction RDA may occured.2) Comparative products analysis(MSn) of lactose and maltose derivated by amino benzene and p-phenylene diamine provided a similar spectrum. The 2-AP and 2-aminoanthraquinone derivatives were similar as well, and the cleavage of C-N bond with a [M+Na]+ ion product m/z 349 did not happen easily, while all of the other derivatives by the reductive amination method produced this peak of much high relative abundance.3) In this research the peak m/z 349 formd by C-N bond cleavage from derivatized lactose is obviously high than that of maltose. In MS2 analysis, lactose derivatized by ABEE, AEC, amino benzene and p-phenylene diamine produced the ion m/z 349 with an abundance up to 100, while the abundance of maltose derivatives is just 20.4) Comparing to the unsaturated ring, saturated ring is not easily to cleave in the form of cross-ring. But it is disparate for the 2-AP and 2-aminoanthraquinone derivatives, whose Y1 ion generated several fragments. The Y1 ion of other derivatives generated few fragments, but Z1 produced more cleavage ions, as Z1 ion was unsaturated ring.5) We concluded from the MS" analysis of the disaccharides derivatized by PMP analysis that the ion type generated can be different if there were different monomer composition and anomeric carbon conformation in oligosaccharide, and there could be much more differences in abundance, such as m/z 521 and m/z 533 in the MS3. We also discovered that PMP-labeled disaccharides were most stable in the derivatives studied in the MSn experiment. For example, as a precursor ion m/z 533 produced much more crossing ring cleavage, and PMP-labeled disaccharides is the same.