Comparison of Collisional and Electron-Based Dissociation Modes for Middle-Down Analysis of Multiply Glycosylated Peptides

Abstract

Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be performed at almost the same scale as proteomics experiments. While collisionally activated dissociation (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of multiply glycosylated peptides. With improvements in instrumentation, electron-activated dissociation (ExD) modes are becoming increasingly prevalent for proteomics experiments and for the analysis of fragile modifications such as glycosylation. While these methods have been applied for glycopeptide analysis in isolated studies, an organized effort to compare their efficiencies, particularly for analysis of multiply glycosylated peptides (termed here middle-down glycoproteomics), has not been made. We therefore compared the performance of different ExD modes for middle-down glycopeptide analyses. We identified key features among the different dissociation modes and show that increased electron energy and supplemental activation provide the most useful data for middle-down glycopeptide analysis. Graphical Abstract.

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

“…Further studies will be needed to explore the utility of AI-ETD for glycopeptides with more than one glycosite, such as those encountered in middle-down and top-down glycoproteomic experiments. 24 Assigning correct glycan modifications for multiply glycosylated peptides poses significant challenges, so we excluded all glycopeptide identifications that harbored more than one glycan in this dataset to ensure higher quality identifications. The middle-down approach can add considerable information to glycoforms and co-occurring glycans, but middle-down methods typically use specifically developed proteolytic and chromatographic methods.…”

“…The middle-down approach can add considerable information to glycoforms and co-occurring glycans, but middle-down methods typically use specifically developed proteolytic and chromatographic methods. Electron-driven dissociation methods have been valuable in middle-down glycoproteomic methods, 24 so it is reasonable to suggest that AI-ETD may prove useful in characterizing multiply glycosylated peptides and proteins as well.…”

“…As such, multiple dissociation strategies (mainly electron-driven dissociation and collision-based methods) are increasingly used to access both glycan and peptide information from intact glycopeptides. 11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.…”

“…As such, multiple dissociation strategies (mainly electron-driven dissociation and collision-based methods) are increasingly used to access both glycan and peptide information from intact glycopeptides. 11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions.…”

Glycoproteome Analysis of Human Serum and Brain Tissue

“…Recent advances in multi-mode fragmentation allow the acquisition of complementary fragmentation spectra on the time-scale of eluting peptide species. (51) The utility of this has been the focus of several recent studies (50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67). We acquired the data using an HCD-triggered EThcD approach relying on detection of an N-acetylglucosamine oxonium ion (4,57,(68)(69)(70)(71)(72)(73)(74).…”