title: Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged, derivatized glycans.
abstract: The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.
authors.affiliation: Department of Chemistry and Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Gennaro
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D010047:Ovalbumin / Q000032:analysis; D011134:Polysaccharides / Q000032:analysis*; D012260:Ribonucleases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: D011134:Polysaccharides; D000509:alpha-Fetoproteins; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B