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Updated: Jun 21, 2026
Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique
Published on: July 19, 2019
Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass
A Cingöz1, F Hugon-Chapuis, V Pichon
1Department of Environmental and Analytical Chemistry, UMR, PECSA, Paris, France.
Abstract:
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).
