Serum protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) appears to be an important diagnostic tool for a whole range of diseases. Sensitivities and specificities obtained with this new technology often seem superior to those obtained with current biomarkers. However, reproducibility and standardization are still problematic. The present review gives an overview of the diagnostic value of protein profiles obtained with SELDI in studies on prostate and ovarian cancer. To identify aspects important for protein profiling, we compare and discuss differences in pre- and post-analytical conditions presented in the literature supplemented with some of our own data. Further progress in protein profiling as a diagnostic tool requires a more comprehensive description of technical details in all future studies.
Background: Knowledge about the presence of intact cardiac troponin T (cTnT) and/or its immunoreactive fragments is of great value for the interpretation of cTnT clearance from the circulation. Until now there has been a lot of controversy about cTnT fragmentation. To provide an answer to this controversy, we investigated fragmentation of cTnT with size-exclusion chromatography (SEC), and confirmed our data using mass spectrometry.
Methods: A highly purified human cTnT standard, characterised using mass spectrometry as a single peak of 34,377 Da and using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a single immunoreactive band (37 kDa), was incubated in serum for 0, 24 and 48h at 37°C and analysed using SEC. A troponin TIC complex standard, used in an earlier study, was also investigated.
Results: We demonstrated that, because of its rod-like shape, the molecular weight of cTnT cannot be estimated from SEC using the molecular weight of globular proteins as a reference. The Stokes radius of intact cTnT was calculated to be 33.7Å. Incubation of both cardiac troponin standards in troponin-free serum resulted in a time-dependent decrease in intact cTnT and a simultaneous increase in smaller immunoreactive fragments (13.4 and 22.4Å).
Conclusions: cTnT has a Stokes radius of 33.7Å. Compared with globular calibrator proteins, intact cTnT elutes earlier than expected based solely on its molecular weight. For non-globular or uncharacterised proteins, Stokes radii should be used for correct interpretation of SEC data. By doing so, we were able to clearly demonstrate cTnT fragments.