title: 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 MS2 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.
authors.affiliation: Process and Product Development, Amgen, Inc., Thousand Oaks, California, USA. xiaoyu-1.chen@novartis.com
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chen
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D008322:Mammals; D008358:Mannose / Q000737:chemistry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D017421:Sequence Analysis / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; D019158:N-Acetylneuraminic Acid; D008358:Mannose; C000219:anthranilamide