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The art of destruction : optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics

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Hinneburg,  Hannes
Daniel Kolarich, Biomolekulare Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society;

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Seeberger,  Peter H.
Peter H. Seeberger - Vaccine Development, Biomolekulare Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society;

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Varón Silva,  Daniel
Daniel Varón Silva, Biomolekulare Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society;

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Kolarich,  Daniel
Daniel Kolarich, Biomolekulare Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society;

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Citation

Hinneburg, H., Stavenhagen, K., Schweiger-Hufnagel, U., Pengelley, S., Jabs, W., Seeberger, P. H., et al. (2016). The art of destruction: optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics. Journal of the American Society for Mass Spectrometry, 27(3), 507-519. doi:10.1007/s13361-015-1308-6.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0029-665A-3
Abstract
In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.