Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis
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 micr...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2012-06, Vol.84 (11), p.5146-5153 |
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| creator | Zhu, Jun Wang, Fangjun Chen, Rui Cheng, Kai Xu, Bo Guo, Zhimou Liang, Xinmiao Ye, Mingliang Zou, Hanfa |
| description | 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. |
| doi_str_mv | 10.1021/ac3000732 |
| format | Article |
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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. 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Chem</addtitle><description>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.</description><subject>Acetonitriles - chemistry</subject><subject>Amino Acid Sequence</subject><subject>Analytical chemistry</subject><subject>Biochemistry</subject><subject>Centrifugation</subject><subject>Chemistry</subject><subject>Chromatography</subject><subject>Chromatography, Liquid</subject><subject>Exact sciences and technology</subject><subject>Glycopeptides - analysis</subject><subject>Glycosylation</subject><subject>Humans</subject><subject>Hydrophilic surfaces</subject><subject>Hydrophobic and Hydrophilic Interactions</subject><subject>Immunoglobulin G - analysis</subject><subject>Limit of Detection</subject><subject>Molecular Sequence Data</subject><subject>Proteolysis</subject><subject>Proteome - analysis</subject><subject>Proteomics</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Trifluoroacetic Acid - chemistry</subject><subject>Trypsin - chemistry</subject><subject>Trypsin - metabolism</subject><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpd0V1rFDEUBuBBFLutXvgHJCCCQkeTMztfl8vYL6gWtF4vZ5MzuymZZJrMCPNL-3fMfthKrwLhyZs3OUnyTvAvgoP4ijLjnJcZvEhmIgeeFlUFL5NZ3MxSKDk_So5DuONcCC6K18kRQF7zuoRZ8tCQHbxuxzUO2lm2CEGHgRT7rqV3nlAOzrMziytDgZ2j1IbYlR1o7fcHXMtu_dQHbdk3vaaw3Txll5Pyrt9oo-VO-5iz1c3Guw4HF0_3mynmei03XaxwytAqdmPTxpmxi1m0NpN0YTL7a9rY4if2Wu3cL7JBD_oPsR_pxdb13g3kOmILi2aKT3iTvGrRBHp7WE-S3-dnt81len1zcdUsrlOcZ_WQgiqhlLLIoYVVRoVsSxCZREIl8oLmGSkUglQ1bwVHWaxQAaBUogAAqsrsJPm0z40N7sf4_GWngyRj0JIbwzLOJ4O6yMU80g_P6J0bfey7UzzOo-J1VJ_3Kn5_CJ7aZe91h36KaOvE8nHa0b4_JI6rjtSj_DfeCD4eAAaJpvVopQ5PLq-LCur5k0MZ_m_1_MK_xvHCaQ</recordid><startdate>20120605</startdate><enddate>20120605</enddate><creator>Zhu, Jun</creator><creator>Wang, Fangjun</creator><creator>Chen, Rui</creator><creator>Cheng, Kai</creator><creator>Xu, Bo</creator><creator>Guo, Zhimou</creator><creator>Liang, Xinmiao</creator><creator>Ye, Mingliang</creator><creator>Zou, Hanfa</creator><general>American Chemical Society</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7TA</scope><scope>7TB</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20120605</creationdate><title>Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis</title><author>Zhu, Jun ; 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Chem</addtitle><date>2012-06-05</date><risdate>2012</risdate><volume>84</volume><issue>11</issue><spage>5146</spage><epage>5153</epage><pages>5146-5153</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><abstract>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.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>22590972</pmid><doi>10.1021/ac3000732</doi><tpages>8</tpages></addata></record> |
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| ispartof | Analytical chemistry (Washington), 2012-06, Vol.84 (11), p.5146-5153 |
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| source | MEDLINE; ACS Publications |
| subjects | Acetonitriles - chemistry Amino Acid Sequence Analytical chemistry Biochemistry Centrifugation Chemistry Chromatography Chromatography, Liquid Exact sciences and technology Glycopeptides - analysis Glycosylation Humans Hydrophilic surfaces Hydrophobic and Hydrophilic Interactions Immunoglobulin G - analysis Limit of Detection Molecular Sequence Data Proteolysis Proteome - analysis Proteomics Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Trifluoroacetic Acid - chemistry Trypsin - chemistry Trypsin - metabolism |
| title | Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis |
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