Hong Yang1, Chenxi Yang1, Taolei Sun1,2. 1. State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, 122 Luoshi Road, Wuhan, 430070, China. 2. School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, 122 Luoshi Road, Wuhan, 430070, China.
Abstract
RATIONALE: Accurate characterization of glycopeptides without a prior glycan cleavage could provide valuable information on site-specific glycosylation, which is critical to reveal the biological functions of protein glycosylation. However, due to the distinct nature of oligosaccharides and ploypeptides, it is usually difficult to effectively fragment glycopeptides in mass spectrometry analysis. METHODS: Here we applied a stepped normalized collisional energy (NCE) approach, which is able to combine fragment ions from three different collision energies, in a hybrid quadrupole orbitrap (Q Exactive Plus) to characterize glycopeptides. A systematic evaluation was firstly performed to find optimal NCE values for the fragmentation of glycan chains and peptide backbones from glycopeptides. Guided by the results of the systematic evaluation, the stepped NCE method was optimized and employed to analyze glycopeptides enriched from human serum. RESULTS: The stepped NCE approach was found to effectively fragment both the glycan chains and peptide backbones from glycopeptides and record these fragments in a single MS/MS spectrum. In comparison with the regular HCD methods, the stepped NCE method identified more glycopeptides with higher scores from human serum samples. CONCLUSIONS: Our studies demonstrate the capability of stepped NCE for the effective characterization of glycopeptides on a large scale.
RATIONALE: Accurate characterization of glycopeptides without a prior glycan cleavage could provide valuable information on site-specific glycosylation, which is critical to reveal the biological functions of protein glycosylation. However, due to the distinct nature of oligosaccharides and ploypeptides, it is usually difficult to effectively fragment glycopeptides in mass spectrometry analysis. METHODS: Here we applied a stepped normalized collisional energy (NCE) approach, which is able to combine fragment ions from three different collision energies, in a hybrid quadrupole orbitrap (Q Exactive Plus) to characterize glycopeptides. A systematic evaluation was firstly performed to find optimal NCE values for the fragmentation of glycan chains and peptide backbones from glycopeptides. Guided by the results of the systematic evaluation, the stepped NCE method was optimized and employed to analyze glycopeptides enriched from human serum. RESULTS: The stepped NCE approach was found to effectively fragment both the glycan chains and peptide backbones from glycopeptides and record these fragments in a single MS/MS spectrum. In comparison with the regular HCD methods, the stepped NCE method identified more glycopeptides with higher scores from human serum samples. CONCLUSIONS: Our studies demonstrate the capability of stepped NCE for the effective characterization of glycopeptides on a large scale.
Authors: Yusi Cui; Ka Yang; Dylan Nicholas Tabang; Junfeng Huang; Weiping Tang; Lingjun Li Journal: J Am Soc Mass Spectrom Date: 2019-07-08 Impact factor: 3.109
Authors: Ruth Barrientos; Carlos Fernández-Galleguillos; Edgar Pastene; Mario Simirgiotis; Javier Romero-Parra; Shakeel Ahmed; Javier Echeverría Journal: Front Pharmacol Date: 2020-05-27 Impact factor: 5.810