Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers.
The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy. volpi@unimo.it
D016667:Bacterial Capsules; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009844:Oligosaccharides; D011135:Polysaccharides, Bacterial; C063323:capsular polysaccharide K4