期刊
ANALYTICAL CHEMISTRY
卷 93, 期 32, 页码 11167-11175出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c01696
关键词
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资金
- National Natural Science Foundation of China [91853131, 81872786, 31971213]
- Guangdong Provincial Key Laboratory of Drug Nonclinical Evaluation and Research [2018B030323024]
- Guangdong Basic, Applied Basic Research Foundation [2019A1515010647]
- Fundamental Research Funds for the Central Universities [19ykzd27]
- Danish National Research Foundation [DNRF107]
Comparison of different dissociation methods for O-glycopeptides showed that ETciD and EThcD outperformed HCD in terms of glycopeptide identification and site localization. Additionally, a pseudo-EThcD strategy combining electron transfer dissociation with paired HCD spectra demonstrated improvements in identification and quantification of isobaric mass tag-labeled O-glycopeptides, leading to the discovery of specific glycosites of GALNT11 in HepG2 cells.
Dissecting site-specific functions of O-glycosylation requires simultaneous identification and quantification of differentially expressed O-glycopeptides by mass spectrometry. However, different dissociation methods have not been systematically compared in their performance in terms of identification, glycosite localization, and quantification with isobaric labeling. Here, we conducted this comparison on highly enriched unlabeled O-glycopeptides with higher-energy collision dissociation (HCD), electron-transfer/collision-induced dissociation (ETciD), and electron transfer/higher-energy collisional dissociation (EThcD), concluding that ETciD and EThcD with optimal supplemental activation resulted in superior identification of glycopeptides and unambiguous site localizations than HCD in a database search by Sequest HT. We later described a pseudo-EThcD strategy that in silica concatenates the electron transfer dissociation spectrum with the paired HCD spectrum acquired sequentially for the same precursor ions, which combines the identification advantage of ETciD/EThcD with the superior reporter ion quality of HCD. We demonstrated its improvements in identification and quantification of isobaric mass tag-labeled O-glycopeptides and showcased the discovery of the specific glycosites of GalNAc transferase 11 (GALNT11) in HepG2 cells.
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