The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics

• Published in: Journal of the American Society for Mass Spectrometry Vol. 27; no. 3; pp. 507 - 519
• Main Authors: Hinneburg, Hannes, Stavenhagen, Kathrin, Schweiger-Hufnagel, Ulrike, Pengelley, Stuart, Jabs, Wolfgang, Seeberger, Peter H., Silva, Daniel Varón, Wuhrer, Manfred, Kolarich, Daniel
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.03.2016; Springer Nature B.V
• Subjects: Amino Acid Sequence; Analytical Chemistry; Animals; Antibiotics; Bioinformatics; Biotechnology; Carbohydrate Sequence; Cattle; Chemistry; Chemistry and Materials Science; Data analysis; Energy of dissociation; Fetuins - chemistry; Fragmentation; Glycan; Glycopeptides; Glycopeptides - analysis; Humans; Immunoglobulins; Immunoglobulins - chemistry; Mass spectrometry; Optimization; Organic Chemistry; Proteomics; Proteomics - methods; Quadrupoles; Quality control; Research Article; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; Glycoproteomics; Q-TOF; Glycopeptide; Collision energy stepping CID; O-glycan; Synthetic glycopeptides; Immunoglobulin; N-glycan
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-015-1308-6

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 Abstract ᅟ