Quantification and site-specific analysis of co-occupied N- and O-glycopeptides.

Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.

False-Positive Glycopeptide Identification via In-FAIMS Fragmentation.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates glycopeptides in the gas phase prior to mass spectrometry (MS) analysis, thus offering the potential to analyze glycopeptides without prior enrichment. Several studies have demonstrated the ability of FAIMS to enhance glycopeptide detection but have primarily focused on N-glycosylation. Here, we evaluated FAIMS for O-glycoprotein and mucin-domain glycoprotein analysis using samples of varying complexity. We demonstrated that FAIMS was useful in increasingly complex samples as it allowed for the identification of more glycosylated species. However, during our analyses, we observed a phenomenon called "in FAIMS fragmentation" (IFF) akin to in source fragmentation but occurring during FAIMS separation. FAIMS experiments showed a 2- to 5-fold increase in spectral matches from IFF compared with control experiments. These results were also replicated in previously published data, indicating that this is likely a systemic occurrence when using FAIMS. Our study highlights that although there are potential benefits to using FAIMS separation, caution must be exercised in data analysis because of prevalent IFF, which may limit its applicability in the broader field of O-glycoproteomics.

Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation.

BACKGROUND: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. OBJECTIVES: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. METHODS: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans. RESULTS: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain. CONCLUSIONS: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.

Mucinomics as the Next Frontier of Mass Spectrometry.

Mucin-domain glycoproteins comprise a class of proteins whose densely O-glycosylated mucin domains adopt a secondary structure with unique biophysical and biochemical properties. The canonical family of mucins is well-known to be involved in various diseases, especially cancer. Despite this, very little is known about the site-specific molecular structures and biological activities of mucins, in part because they are extremely challenging to study by mass spectrometry (MS). Here, we summarize recent advancements toward this goal, with a particular focus on mucin-domain glycoproteins as opposed to general O-glycoproteins. We summarize proteolytic digestion techniques, enrichment strategies, MS fragmentation, and intact analysis, as well as new bioinformatic platforms. In particular, we highlight mucin directed technologies such as mucin-selective proteases, tunable mucin platforms, and a mucinomics strategy to enrich mucin-domain glycoproteins from complex samples. Finally, we provide examples of targeted mucin-domain glycoproteomics that combine these techniques in comprehensive site-specific analyses of proteins. Overall, this Review summarizes the methods, challenges, and new opportunities associated with studying enigmatic mucin domains.