Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Ion chromatography coupled with mass spectrometry (IC-MS): an innovative tool for the characterization of the sulfated O-linked glycans (#816)

Zoltan Szabo 1 , Neil G Rumachik 2 , Jim Thayer 2 , Gina Tan 3 , Rosa Viner 3 , Andreas Huhmer 3 , Jeff Rohrer 2 , Yan Liu 2 , Dietmar Reusch 4 , Christopher A Pohl 2
  1. Thermo Fisher Scientific, South San Francisco, CA
  2. Thermo Fisher Scientific, Sunnyvale, CA, United States
  3. Thermo Fisher Scientific, San Jose, CA
  4. Roche Diagnostics GmbH, Penzberg, Germany

High Performance Anion Exchange Chromatography (HPAE) has long been used to separate ions and polar molecules. Here, we demonstrate that HPAE can be coupled to an Orbitrap™ mass spectrometer (IC-MS) to facilitate the separation and sequencing of sulfated O-glycan alditols. Porcine gastric mucin (type III) harbors high levels of sulfated but fewer sialylated glycans, and after release and rapid purification, these O-linked glycans were directly injected and analyzed. However, to analyze bovine submaxillary mucin, which harbors fewer sulfated but more sialylated O-glycans, a new workflow based on weak anion exchange (WAX) fractionation was necessary to minimize ion-suppression by the sialylated glycans. Using a post column splitter, 60% of the flow was desalted with an electrochemically regenerated desalter prior to introduction into the mass spectrometer. Due to the ability of HPAE to resolve isomers, 27 sulfated O-linked glycans were identified from Porcine gastric mucin (type III), and 9 from bovine submaxillary mucin. Using the data-dependent mode of the Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer, highly informative MS2 spectra enabled confident identification of the separated species.