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.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.
Highlights
Protein glycosylation is one of the most common posttranslational modifications [1]
Compared with alternative fragmentation techniques such as electron transfer dissociation (ETD), we found collision energy stepping collision induced dissociation (CID) to be more robust in delivering useful data within a data-dependent LC-electrospray ionization (ESI) tandem MS experiment, in particular if precursors with high m/z values were selected
Under the conditions applied in this study, the most informative glycopeptide spectra were obtained when collision energies of 55 eV at 700 m/z to 124 eV at 1800 m/z were applied in a m/z dependent manner
Summary
Protein glycosylation is one of the most common posttranslational modifications [1]. The vast majority of Electronic supplementary material The online version of this article (doi:10. 1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.H. The vast majority of Electronic supplementary material The online version of this article 1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users. H. Hinneburg et al.: The Art of Glycopeptide Destruction have been shown to be crucial for influencing the physicochemical and functional properties of their respective protein carriers. In IgE, the site-specific presence of an oligomannose-type N-glycan has been shown to be necessary for initiating anaphylaxis [11]. These examples illustrate that knowledge about site-specific glycosylation is an important prerequisite for studying the functional impact of protein glycosylation
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