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

Intact glycopeptide analysis by trapped ion mobility tandem mass spectrometry    (#593)

Hans JCT Wessels 1 , Melissa Bärenfänger 1 , Merel Post 1 , Kristina Marx 2 , Pierre-Olivier Schmit 3 , Markus Lubeck 2 , Alain Van Gool 1 , Dirk Lefeber 1 , Jaran Jainhuknan 3
  1. Radboud UMC, Nijmegen, Netherlands
  2. Bruker Daltonik GmbH, Bremen, Germany
  3. Bruker France, Wissembourg, France


Recent introduction of parallel accumulation serial fragmentation (PASEF) on a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro) provides unique possibilities for comprehensive glycopeptide profiling in complex samples such as blood plasma. Glycosylated peptides are known for their poor ionization efficiency during electrospray ionization which we addressed by providing dopant enriched nitrogen gas into an axial desolvation vacuum assisted electrospray ionization source (CaptiveSpray nanoBooster). Here, we evaluated the effects of different dopants for glycopeptide analysis by ion mobility mass spectrometry and PASEF.



Glycopeptides from model glycoproteins and blood plasma were separated by reversed phase liquid chromatography (nanoElute, Bruker Daltonics) with online analysis in a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro, Bruker Daltonics) using acetonitrile and primary alcohols as nitrogen gas dopants in the ESI source (CaptiveSpray nanoBooster). Data analysis was performed in DataAnalysis (Bruker Daltonics), ProteinScape/GlycoQuest (Bruker Daltonics), PEAKS studio (Bioinformatics Solutions), and by in-house developed scripts.



Use of dopants in nanoflow ESI on the timsTOF Pro platform requires significant adjustment of the TIMS vacuum conditions and prohibits the use of fluorinated phosphazene calibrants due to signal loss. Alternatively, Poly-DL-Alanine can be used as calibrant which also offers additional data points for TIMS calibration. Acetonitrile and primary alcohol dopants significantly enhance the ionization efficiency of glycopeptides in ESI compared to air or nitrogen gas. Acetonitrile supercharges glycopeptides whereas primary alcohols subcharge glycopeptides. Interestingly, primary alcohols also reduce the number of charge states per ion which simplifies the ion mixture prior to spectrometric analysis. At present, we analyze for each dopant the ion mobility characteristics as well as PASEF performance for glycan- and peptide-moiety characterization.



This work present the first application and evaluation of ESI dopants for enhanced glycopeptide characterization by trapped ion mobility mass spectrometry and PASEF.​