Optimal Dissociation Methods Differ for N- and O-glycopeptides

Abstract

<p><a>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 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, indicating that ETD-based methods are not required for routine N-glycoproteomics. 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.</a></p>

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

“…In a study of tryptic glycopeptides enriched with wheat germ agglutinin from human urine using this ionization method, Darula et al ( 2019) identified new O-acetyl-sialic acid (Neu5,9Ac 2 , 195) on the galactose residues of core-1 (196), and core-2 (197) O-glycans. , Malaker, Driessen, et al (2020) have compared the advantages and disadvantages of conventional HCD, stepped collision energy (sceHCD), ETD, and electron transfer dissociation with HCD supplemental activation (EThcD) for the analysis of intact N-and O-glycopeptides. For N-glycopeptides, HCD and sceHCD generated similar numbers of identifications, although sceHCD generally provided higher quality spectra.…”

Maximizing glycoproteomics results through an integrated PASEF workflow

“…Other software tools that take glycan fragmentation into account can benefit from collision energy stepping. [24][25]33 We therefore provide the optimized instrument method both with and without TIMS stepping in the ProteomeXchange repository under the identifier PXD047898. Data was visualized using Python 3.10.10 with the packages Matplotlib 3.7.1, Scipy 1.10.1, Numpy 1.24.3 and Pandas 1.5.3.…”

“…Potential solutions are the use of advanced fragmentation techniques like electron-based dissociation (ExD), that produce c-and z-type peptide fragment ions while retaining the intact glycan moiety. [25][26] However, these techniques are not widely available. More commonly used CID instruments can overcome this obstacle by using higher collision energies to increase the yield of peptide fragment ions or performing CID stepping to fragment the glycan moiety at lower collision energies and the peptide moiety at high collision energies.…”