{"title":"利用免疫提取、消化和液相色谱-质谱法对血浆中目标蛋白进行全面在线分析。","authors":"A Cingöz, F Hugon-Chapuis, V Pichon","doi":"10.1016/j.jchromb.2009.07.032","DOIUrl":null,"url":null,"abstract":"<p><p>A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).</p>","PeriodicalId":520661,"journal":{"name":"Journal of chromatography. B, Analytical technologies in the biomedical and life sciences","volume":" ","pages":"213-21"},"PeriodicalIF":0.0000,"publicationDate":"2010-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jchromb.2009.07.032","citationCount":"36","resultStr":"{\"title\":\"Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry.\",\"authors\":\"A Cingöz, F Hugon-Chapuis, V Pichon\",\"doi\":\"10.1016/j.jchromb.2009.07.032\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).</p>\",\"PeriodicalId\":520661,\"journal\":{\"name\":\"Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\",\"volume\":\" \",\"pages\":\"213-21\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2010-01-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/j.jchromb.2009.07.032\",\"citationCount\":\"36\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://doi.org/10.1016/j.jchromb.2009.07.032\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2009/8/3 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of chromatography. B, Analytical technologies in the biomedical and life sciences","FirstCategoryId":"1","ListUrlMain":"https://doi.org/10.1016/j.jchromb.2009.07.032","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2009/8/3 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry.
A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).