Journal
JOURNAL OF PROTEOME RESEARCH
Volume 19, Issue 8, Pages 3286-3301Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.0c00218
Keywords
glycoproteomics; N-glycopeptides; O-glycopeptides; tandem MS; fragmentation; electron transfer dissociation; stepped collision energy high-energy collisional dissociation; ETD; EThcD; sceHCD
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Funding
- Howard Hughes Medical Institute
- National Institute of Health (NIH) [R01 CA200423]
- NIH [U01-CA207702]
- NIH F32 Postdoctoral Fellowship [F32-GM126663-01]
- NIH Predoctoral to Postdoctoral Transition Award [K00 CA21245403]
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Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N-glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.
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