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Xinmiao Liang - One of the best experts on this subject based on the ideXlab platform.
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tio2 simultaneous enrichment on line Deglycosylation and sequential analysis of glyco and phosphopeptides
Frontiers in Chemistry, 2021Co-Authors: Xiaofei Zhang, Cheng Chen, Xuefang Dong, Han Zhou, Xinmiao LiangAbstract:Reversible protein glycosylation and phosphorylation tightly modulate important cellular processes and are closely involved in pathological processes in a crosstalk dependent manner. Because of their significance and low abundances of glyco- and phosphopeptides, several strategies have been developed to simultaneously enrich and co-elute glyco- and phosphopeptides. However, the co-existence of deglycosylated peptides and phosphopeptides aggravates the mass spectrometry analysis. Herein we developed a novel strategy to analyze glyco- and phosphopeptides based on simultaneous enrichment with TiO2, on-line Deglycosylation and collection of deglycosylated peptides, and subsequent elution of phosphopeptides. To optimize on-line Deglycosylation conditions, the solution pH, buffer types and concentrations, and Deglycosylation time were investigated. The application of this novel strategy to 100 μg mouse brain resulted in 355 glycopeptides and 1,975 phosphopeptides, which were 2.5 and 1.4 folds of those enriched with the reported method. This study will expand the application of TiO2 and may shed light on simultaneously monitoring protein multiple post-translational modifications.
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Table5_TiO2 Simultaneous Enrichment, On-Line Deglycosylation, and Sequential Analysis of Glyco- and Phosphopeptides.xlsx
'Frontiers Media SA', 2021Co-Authors: Cheng Chen, Xiaofei Zhang, Xuefang Dong, Han Zhou, Xinmiao LiangAbstract:Reversible protein glycosylation and phosphorylation tightly modulate important cellular processes and are closely involved in pathological processes in a crosstalk dependent manner. Because of their significance and low abundances of glyco- and phosphopeptides, several strategies have been developed to simultaneously enrich and co-elute glyco- and phosphopeptides. However, the co-existence of deglycosylated peptides and phosphopeptides aggravates the mass spectrometry analysis. Herein we developed a novel strategy to analyze glyco- and phosphopeptides based on simultaneous enrichment with TiO2, on-line Deglycosylation and collection of deglycosylated peptides, and subsequent elution of phosphopeptides. To optimize on-line Deglycosylation conditions, the solution pH, buffer types and concentrations, and Deglycosylation time were investigated. The application of this novel strategy to 100 μg mouse brain resulted in 355 glycopeptides and 1,975 phosphopeptides, which were 2.5 and 1.4 folds of those enriched with the reported method. This study will expand the application of TiO2 and may shed light on simultaneously monitoring protein multiple post-translational modifications.
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DataSheet1_TiO2 Simultaneous Enrichment, On-Line Deglycosylation, and Sequential Analysis of Glyco- and Phosphopeptides.pdf
'Frontiers Media SA', 2021Co-Authors: Cheng Chen, Xiaofei Zhang, Xuefang Dong, Han Zhou, Xinmiao LiangAbstract:Reversible protein glycosylation and phosphorylation tightly modulate important cellular processes and are closely involved in pathological processes in a crosstalk dependent manner. Because of their significance and low abundances of glyco- and phosphopeptides, several strategies have been developed to simultaneously enrich and co-elute glyco- and phosphopeptides. However, the co-existence of deglycosylated peptides and phosphopeptides aggravates the mass spectrometry analysis. Herein we developed a novel strategy to analyze glyco- and phosphopeptides based on simultaneous enrichment with TiO2, on-line Deglycosylation and collection of deglycosylated peptides, and subsequent elution of phosphopeptides. To optimize on-line Deglycosylation conditions, the solution pH, buffer types and concentrations, and Deglycosylation time were investigated. The application of this novel strategy to 100 μg mouse brain resulted in 355 glycopeptides and 1,975 phosphopeptides, which were 2.5 and 1.4 folds of those enriched with the reported method. This study will expand the application of TiO2 and may shed light on simultaneously monitoring protein multiple post-translational modifications.
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n glycosylation site analysis of proteins from saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography based enrichment parallel Deglycosylation and mass spectrometry
Journal of Proteome Research, 2014Co-Authors: Liwei Cao, Zhimou Guo, Aijin Shen, Yunu Guo, Xinmiao LiangAbstract:N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel Deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf Deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Jun Zhu, Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Zhimou Guo, Hanfa ZouAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
Kai Cheng - One of the best experts on this subject based on the ideXlab platform.
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highly efficient release of glycopeptides from hydrazide beads by hydroxylamine assisted pngase f Deglycosylation for n glycoproteome analysis
Analytical Chemistry, 2015Co-Authors: Kai Cheng, Junfeng Huang, Hao Wan, Yating Yao, Jiawei Mao, Jin ChenAbstract: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.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Jun Zhu, Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Zhimou Guo, Hanfa ZouAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Bo Xu, Mingliang YeAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 22...
Fangjun Wang - One of the best experts on this subject based on the ideXlab platform.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Jun Zhu, Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Zhimou Guo, Hanfa ZouAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Bo Xu, Mingliang YeAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 22...
Hanfa Zou - One of the best experts on this subject based on the ideXlab platform.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Jun Zhu, Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Zhimou Guo, Hanfa ZouAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
Mingliang Ye - One of the best experts on this subject based on the ideXlab platform.
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centrifugation assisted microreactor enables facile integration of trypsin digestion hydrophilic interaction chromatography enrichment and on column Deglycosylation for rapid and sensitive n glycoproteome analysis
Analytical Chemistry, 2012Co-Authors: Fangjun Wang, Kai Cheng, Xinmiao Liang, Rui Chen, Bo Xu, Mingliang YeAbstract:Sample handling procedures including protein digestion, glycopeptide enrichment, and Deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and Deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 22...