Structural characterization of sialylated glycoforms of H. influenzae by electrospray mass spectrometry: fragmentation of protonated and sodiated O‐deacylated lipopolysaccharides

Abstract

Sialylated lipopolysaccharide (LPS) glycoforms from Haemophilus influenzae were characterized by tandem mass spectrometry using a new generation hyphenated mass spectrometer which combines a triple quadrupole and a linear ion trap (Q-Trap). The fragmentation of both protonated and sodiated molecular ions from O-deacylated LPS (LPS-OH) obtained in MS(2) experiments in the positive mode was studied. The MS(2) spectra of protonated ions provided unambiguous evidence for the presence and sequence of sialylated lactosamine present in lacto-N-neotetraose oligosaccharide extensions but not for sialyl-lactose structures whilst fragmentation of sodiated adducts, [M+Na](+), afforded information diagnostic of mono- and disialylated lactose extensions. To study this we used a highly sialylated LPS from a H. influenzae strain capable of sialyl-lactose expression only. We then applied the method to the H. influenzae genome strain, Rd, in which glycoforms containing both sialyl-lactose and sialyl-lacto-N-neotetraose were detected from diagnostic B-ions at m/z 638.2 ([Neu5Ac(1) Hex(2)+Na](+)) and 657.2 ([Neu5Ac(1) Hex(1) HexNAc(1)+H](+)). Unique fragmentation patterns provided the locations and sequences of these oligosaccharide extensions. This is the first time both sialylated lactose and sialylated lacto-N-neotetraose units have been detected and characterized by tandem mass spectrometry in the same molecule. This methodology is of general applicability for determination of common sialylated oligosaccharide extension in bacterial LPS.

Mass spectrometry for protein sialoglycosylation

“…Mass spectrometric analysis of intact proteins is a method to directly assess global compositional properties of carbohydrate moieties. It is worth mentioning that sialylation is also an important feature of glycolipids such as gangliosides, lipooligosaccharides (LOS) and lipopolysaccharides (LPS) . Further improvement in ganglioside analysis includes negative‐ion electrospray ionization (ESI)‐MS, matrix‐assisted laser desorption/ionization (MALDI)‐MS, MS imaging, and ion mobility (IM)‐MS .…”

Separation and characterization of lipopolysaccharide related compounds by HPLC/post-column fluorescence derivatization (HPLC/FLD) and capillary zone electrophoresis/mass spectrometry (CZE/MS)

“…As a volatile running buffer, ammonium formate and acetate were thought to be compatible with the ion trap-mass spectrometry (IT-MS) analysis of the fractions directly from the capillary. Although morpholine buffer has been reported as appropriate in some papers [15,16], 10 mM ammonium formate and ammonium acetate had superior sensitivities at the IT-MS detector.…”

“…Thus capillary zone electrophoresis/mass spectrometry (CZE/MS) was investigated as an alternative method. CZE/MS has been previously reported for the analyses of the deacylated derivatives of LPS from Haemophilus influenzae [15,16].…”

Capillary electrophoresis of acidic oligosaccharides from human milk

“…The role of CE-MS in biological analysis has been increasing, allowing rapid identification and quantification of target analytes present at trace levels in biological matrix and providing structural characterization of closely related complex biomolecules. For example, CE-MS has been employed to map glycoforms of bacteria [70,71], to separate and characterize glycoform isomers [72,73], and to detect trace levels of carbohydrates [74][75][76]. However, a method for CE-MS of HMOS has yet to be published.…”