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Enhancing glycopeptide annotation and glycan localization using the EAD implementation of hot electron capture dissociation through a multiplexed parallel reaction monitoring design of experiment

ABSTRACT: We present an optimized electron activated dissociation (EAD) methodology for liquid chromatography-tandem mass spectrometry (LC-MS/MS) based characterization of N- and O-glycopeptides. Recombinant human erythropoietin (rhEPO) was used as a model glycoprotein in this study. Applying a full factorial design of experiment (DoE) approach on the ZenoTOF 7600 instrument, we first optimized LC-MS parameters (i.e., ion spray voltage, ion source temperature, and active gradient time) to enhance glycopeptide ionization efficiency while reducing in-source fragmentation (ISF). Then, another DoE was performed for EAD parameter optimization. Multiplexed parallel reaction monitoring of one glycoform of each glycopeptide was performed to efficiently and comprehensively optimize the electron beam current, reaction time, and electron kinetic energy of the EAD set-up. Finally, the optimized EAD parameters were successfully applied in data-dependent acquisition (DDA) mode for the untargeted analysis of tryptic digests of rhEPO. Byonic and Mascot softwares were used to evaluate the potential of our optimized EAD setup against collision induced dissociation (CID), confirming that with EAD we improved glycan localization confidence while with CID a superior number of glycoforms were identified although with less confident glycan assignment.

INSTRUMENT(S):

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus)

SUBMITTER: Maarten Dhaenens

LAB HEAD: Maarten Dhaenens

PROVIDER: PXD063177 | Pride | 2025-10-23

REPOSITORIES: Pride

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Source:

			Action	DRS
	221123_EPO_Crap.fasta	Fasta
	230118_01_Tryptic_20min_4500_200_26B.zip	Other
	230118_02_Tryptic_20min_4500_200_26B.zip	Other
	230118_03_Tryptic_20min_4500_200_26B.zip	Other
	230118_13_Tryptic_15min_3500_150_26B.zip	Other

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Enhancing glycopeptide annotation and glycan localization using the EAD implementation of hot electron capture dissociation through a multiplexed parallel reaction monitoring design of experiment

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