This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Glycoconjugate glycans consist of mixtures of variants, known as glycoforms, on a common core structure. These variants arise as a result of biosynthetic events under complex regulation. One of the challenges in mass spectral analysis of glycoconjugate glycans is that the ion signals corresponding to a given oligosaccharide composition may be produced by a mixture of structural isomers. Ion mobility spectrometry (IMS) entails passing ions through a mobility cell operated at elevated pressure, relative to vacuum. For a given charge state, the mobility time increases with the collisional cross section of the ions. The goal of this work is to determine the extent to which carbohydrate isomers may be resolved using ion mobility. Ion mobility spectra were acquired using a modified Waters QTOF Premier equipped with a traveling wave ion guide operated at 1 mbar. The ion guided consisted of 122 parallel plates, each with a 2.5 mm orifice and center-to-center spacing of 1.5 mm. Oligosaccharides were dissolved at 1 pmol/?L in 10% isopropanol and infused into the electrospray source at 5 ?L/min. Each 15-ms ion mobility spectrum consisted of a series of 200 75-?s TOF scans. Approximately 360 ion mobility spectra were summed to produce scans displayed on the data system with a 5.4 s repeat, and 20-30 such scans were summed to produce the final spectra. The following compound classes were studied: native glycosaminoglycan disaccharides, native and permethylated milk oligosaccharides, and native and permethylated high mannose N-linked oligosaccharides. The [M-H]- ions generated from the pair ?HexA(?1,3)GalNAc and ?HexA(?1,4)GlcNAc produced the same ion mobility. A series of five isomers of the composition (?HexA)(HexNAc)(SO3) produced subtle differences in mobility for [M-H]- ions and no differences for the [M-2H]2- ions. A series of three isomers of composition (HexA)(HexNAc)(SO3)2 produced distinct mobility profiles that differentiated isomers for [M-H]- and [M(Na)-H]- , [M-2H]2-, and [M(Na)-2H]2- ions. The mobility differences corresponded to one or two 75-usec TOF scans. Milk oligosaccharides LNT and LNnT, tetrasaccharides differing by the Gal-GlcNAc linkage, produced mobility traces differing by two TOF scans for native [M-H]-, native [M+Na]+ and permethylated [M+Na]+ ions. The sialylated forms of these glycans, LST-a and LST-d, produced mobility traces that differed by two TOF scans for native [M-H]-, [M(Na)+H]+, and [M(Na)+Na]+ . The permethylated forms of the LST glycans produced identical mobility traces. Lewis oligosaccharides (LeX and LeA) produced mobility traces differing by one TOF scan for native [M-H]- and permethylated [M+Na]+ ions. The sialylated forms (sialyl LeX and sialyl LeA) produced identical mobility traces for native [M-H]- and permethylated [M+Na]+ ions. High mannose oligosaccharides released from ribonuclease B consist of a series of glycoforms containing 5-9 mannose residues. The glycoforms were all clearly resolved on the basis of composition in the mobility traces for native [M-H]- and [M+Na]+ and permethylated [M+Na]+. The (GlcNAc)2(Man)7 glycoform exists as a mixture of positional isomers. These isomers were not resolved in any of the experiments. Further experiments are underway to explore conditions that may enable isomer resolution of these larger glycans.
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