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 clue 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 I laboratory-made IMER based on pepsin The whole on-line device (ISIMER-LC-MS/MS) allowed the quantification of cytochrome c from 8 5 pmol to 1 7 nmol in buffer medium Finally. this device was applied to the analysis of only 85 pmol of cytochrome c from plasma with a RSD value lower than 10%(n=3) (C) 2009 Elsevier B.V. All rights reserved