title: Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.
abstract: BACKGROUND
  Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively.
  
  
  METHODS
  N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer.
  
  
  RESULTS
  Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified.
  
  
  CONCLUSIONS
  One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels.
authors.affiliation: Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090, Vienna, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Michael
authors.affiliation: Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090, Vienna, Austria.
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Rizzi
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006108:Graphite / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D012794:Sialic Acids / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D000814:Aniline Compounds; D002247:Carbon Isotopes; D011134:Polysaccharides; D012794:Sialic Acids; D002244:Carbon; D006108:Graphite; C023650:aniline