Quantification of TAFI in human plasma by LC-MS/MS
M. Rauh, S. Weiß, K. Heußner, M. Chada, J. Zierk, W. Rascher, H.-G. Topf (Erlangen, Germany)
Time: 17:15 - 18:15
Objective: Thrombin activatable fibrinolysis inhibitor (TAFI) is supposed to play a role in thrombotic disorders as well as in inflammatory diseases. Further, a defective activation of TAFI might be related to the severity of bleeding disorders like hemophilia A. To measure plasma TAFI concentrations, different methods have been developed based on antigen determination and measurement of TAFI activity after quantitative conversion of the proenzyme. These assays have major drawbacks. They are lacking standardization and show different analytical sensitivity to the TAFI cleavage products. Mass spectrometry has the potential to overcome some of observed assay limitations. In the case of proteins, a specific tryptic peptide can be selected as a stoichiometric representative of the protein from which it is cleaved.
Methods: Our aim was to use LC-MS/MS to quantify TAFI activity by Liquid Chromatography/Electrospray Ionization Mass Spectrometry. We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of TAFI in human plasma by LC‐MS/MS using proteotypic peptides and corresponding stable isotope‐labeled peptides as internal standards.
Results: With isotopic labeled peptides as matched internal standards, we established a quantitative assay for the proteotypic peptide DTGTYGFLLPER and compared it to a commercially available activity assay (r = 0.95) and proTAFI immunoassay (r= 0.96). We have tested several methods of sample processing and established a fast and sensitive chromatographic assay. The validated preanalytical protocol enables a reliable analysis of TAFI antigen in human plasma without depletion.
Conclusion: The assay can be very useful in future research into the identification of pathological conditions where TAFI is generated and would be helpful for standardization .