Systematic Evaluation of Fragmentation Methods for Unlabeled and Isobaric Mass Tag-Labeled O-Glycopeptides
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Systematic Evaluation of Fragmentation Methods for Unlabeled and Isobaric Mass Tag-Labeled O-Glycopeptides. / Mao, Yang; Wang, Shengjun; Zhao, Yuanqi; Konstantinidi, Andriana; Sun, Lingyu; Ye, Zilu; Vakhrushev, Sergey Y.
In: Analytical Chemistry, Vol. 93, No. 32, 2021, p. 11167-11175.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Systematic Evaluation of Fragmentation Methods for Unlabeled and Isobaric Mass Tag-Labeled O-Glycopeptides
AU - Mao, Yang
AU - Wang, Shengjun
AU - Zhao, Yuanqi
AU - Konstantinidi, Andriana
AU - Sun, Lingyu
AU - Ye, Zilu
AU - Vakhrushev, Sergey Y.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - ELECTRON-TRANSFER DISSOCIATION
KW - CELL-ADHESION
KW - GLYCOSYLATION
KW - PEPTIDE
KW - QUANTIFICATION
KW - IDENTIFICATION
KW - PROTEOMICS
KW - NOTCH
KW - ITRAQ
KW - ETD
U2 - 10.1021/acs.analchem.1c01696
DO - 10.1021/acs.analchem.1c01696
M3 - Journal article
C2 - 34347445
VL - 93
SP - 11167
EP - 11175
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 32
ER -
ID: 278038516