Poster Presentation 11th Australian Peptide Conference 2015

A systematic investigation of CID Q-TOF collision energies for complete identification of glycopeptides by mass spectrometry (#111)

Maja Christiansen 1 , Hannes Hinneburg 2 3 , Kathrin Stavenhagen 4 , Ulrike Schweiger-Hufnagel 5 , Stuart Pengelley 5 , Peter H Seeberger 2 3 , Daniel Varón Silva 2 , Manfred Wuhrer 4 , Daniel Kolarich 2
  1. Bruker Pty Ltd, Alexandria, NSW, Australia
  2. Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, GERMANY
  3. Free University Berlin, Berlin, GERMANY
  4. VU University Amsterdam, Amsterdam, NETHERLANDS
  5. Bruker Daltonik GmbH, Bremen, GERMANY

Introduction

In most naturally occurring glycoproteins, pools of glycans are attached to one or more glycosylation sites. The analysis of each individual site is challenging and requires the analysis of glycopeptides. For this, quadrupole time of flight (QTOF) mass spectrometers (MS) with high mass accuracy, fast duty cycles, and high m/z range are highly suited due to the usability of multiple collision energies. The systematic investigation of optimum collision energies for the glycan and the peptide part of glycopeptides as well as the software supported data interpretation is presented in this approach.

Material & Methods
Synthetic N-glycopeptides were analysed on a QTOF MS instrument (impact II) with CaptiveSpray nanoBoosterÔ (Bruker Daltonics). Tryptically digested standard glycoproteins (fetuin, antibodies) were separated by nano LC before MS analysis. Collision energies were systematically varied. Glycopeptide spectra were detected and the peptide masses were determined automatically (ProteinScape 4.0). Glycan structures were identified using the integrated GlycoQuest search engine, and for peptide identification Mascot (Matrix Sciences) was used.

Results
The fragmentation parameters on QTOF instruments were systematically investigated using synthetic glycopeptides and glycopeptide mixtures. This allowed identifying conditions resulting in maximum sequence information on both, peptide and glycan parts of glycopeptides. The energies required for optimal glycan fragmentation were found to be clearly below the ones necessary for the peptide part. Nevertheless, the data showed a narrow energy range of +/- 5 eV produced spectra that resulted in the highest scores.

The optimized parameters were successfully applied on digested monoclonal antibodies and complex glycoprotein mixtures, which allowed the identification of complete N- and O-glycopeptides. This improved methodology is particularly useful in the fields of glycoproteomics research as well as biopharmaceutical development and quality control.

Conclusion
Applying different collision energies on a QTOF instrument, the peptide and the glycan part of glycopeptides were fragmented. This allowed the identification of complete glycopepides.