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).
Cingöz
A
A
Department of Environmental and Analytical Chemistry, UMR, PECSA, Paris, France.
Hugon-Chapuis
F
F
Pichon
V
V
D055910:Antibodies, Immobilized / Q000378:metabolism; D019149:Bioreactors; D002853:Chromatography, Liquid / Q000379:methods*; D045304:Cytochromes c / Q000097:blood* / Q000276:immunology; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007163:Immunosorbent Techniques; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D055910:Antibodies, Immobilized; D055767:Immobilized Proteins; D010446:Peptide Fragments; D045304:Cytochromes c; D010434:Pepsin A