title: Highly Efficient Release of Glycopeptides from Hydrazide Beads by Hydroxylamine Assisted PNGase F Deglycosylation for N-Glycoproteome Analysis.
abstract: Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Junfeng
authors.initials: J
authors.lastname: Huang
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology , Shanghai 200237, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wan
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Yating
authors.initials: Y
authors.lastname: Yao
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Jinan
authors.initials: J
authors.lastname: Li
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Mao
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.|University of Chinese Academy of Sciences , Beijing, China , 100049.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology , Shanghai 200237, China.
authors.forename: Weibing
authors.initials: W
authors.lastname: Zhang
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, China , 116023.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D006020:Glycopeptides / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D006834:Hydrazines / Q000737:chemistry*; D019811:Hydroxylamine / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D006834:Hydrazines; D020543:Proteome; D019811:Hydroxylamine; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase