Gas-phase oligosaccharide nonreducing end (GONE) sequencing and structural analysis by reversed phase hplc/mass spectrometry with polarity switching

Abstract

Here we describe a technique to obtain all the N-linked oligosaccharide structures from a single reversed-phase (RP) HPLC run using on-line tandem MS in both positive and negative ion modes with polarity switching. Oligosaccharides labeled with 2-aminobenzamide (2AB) were used because they generated good ionization efficiency in both ion polarities. In the positive ion mode, protonated oligosaccharide ions lose sugar residues sequentially from the nonreducing end with each round of MS fragmentation, revealing the oligosaccharide sequence from greatly simplified tandem MS spectra. In the negative ion mode, diagnostic ions, including those from cross-ring cleavages, are readily observed in the MS 2 spectra of deprotonated oligosaccharide ions, providing detailed structural information, such as branch composition and linkage positions. Both positive and negative ion modes can be programmed into the same LC/MS experiment through polarity switching of the MS instrument. The gas-phase oligosaccharide nonreducing end (GONE) sequencing data, in combination with the diagnostic ions generated in negative ion tandem MS, allow both sequence and structural information to be obtained for all eluting species during a single RP-HPLC chromatographic run. This technique generates oligosaccharide analyses at high speed and sensitivity, and reveals structural features that can be difficult to obtain by traditional methods. . Proper structural determination is thus critical to the understanding of the biological role of carbohydrates. Although there are fewer monosaccharide building blocks in N-linked glycans than there are amino acid moieties in proteins, the combination of multiple branchings and linkages add greater complexity to the glycan structures. Such structural diversity also adds to the difficulty of structural characterization.Many analytical tools are now available for oligosaccharide characterization, such as high field NMR, liquid chromatography, capillary electrophoresis, and mass spectrometry. Although unambiguous oligosaccharide structural assignments can be made by NMR [2][3][4][5], this technique often requires large quantities of highly purified material. Typically, more than 50 mg of glycoprotein and extensive preparative fractionation are required to resolve oligosaccharide structures of relatively low abundance. A more common characterization approach utilizes sets of specific exoglycosidases, which cleave terminal monosaccharides from the nonreducing end, followed by chromatographic or MS analysis [6][7][8]. In this approach, the sequence of the oligosaccharide chain is revealed, either through sequential removal of monosaccharides from the chain upon successive enzyme treatment, or from the oligosaccharide ladder generated upon treatment with an array of enzymes. Linkage positional information and anomericity of the terminal monosaccharide can also be obtained, based on the specificity of the exoglycosidases used. Unfortunately, this classic sequencing technique is often labor intensive, time consuming, and...

Reversed-phase separation methods for glycan analysis

“…Labeling with AA provides at least two times more sensitivity in fluorescence detection as compared with labeling with AB [79]. In addition, AB and AA can both be used in positive and negative ion mode MS [32, 38, 81]. The labeling procedures for AA and AB labels were optimized by Bigge et al [87] in the 1990s.…”

“…Chen and Flynn [80] also performed reversed-phase separation of AB-labeled N -glycans, and found a slightly different elution order of the different types of glycans. They described that fucosylated sialylated glycans are eluted first from the column, followed by high-mannose glycans and neutral complex glycans [80, 81]. The difference between the elution order described by Higel et al and this elution order might be due to differences in the columns, eluents, and gradients used, but this was not investigated.…”

Glycan labeling strategies and their use in identification and quantification

“…For fluorescence detection in RP-HPLC, the most commonly applied tags are PA [93, 178] and 2-AB [179, 180]. Together with HILIC and anion-exchange chromatography, RP-HPLC with fluorescence detection is used in multidimensional mapping strategies for structural assignment of PA-labeled N -glycans [93].…”

“…Moreover, both 2-AB-labeled and 2-AA-labeled N -glycans can be analyzed by RP-nanoLC-MS/MS, and separations of structural isomers have been demonstrated [83, 179, 180, 183]. Chen and Flynn [180] have impressively demonstrated the separation power of RP-HPLC of 2-AB-labeled glycans, and have combined this with both positive-mode and negative-mode multistage tandem MS on an ion trap mass spectrometer for structural assignment (Fig. 7).…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…5). 17,23,59,102,104 2-AP is still regarded as being one of the most suitable labeling reagents for the displacement mode of RP-HPLC, because the low hydrophobicity of 2-AP means that it only has a small effect on the retention times, which enables the emphasis to be on the glycan structures.…”