Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
The medical field is slowly evolving from traditional views, such as fixed dose for all patients, to taking in aspects of variance between individuals for both treatments and diagnostics. For further modernisation of the medical field should the way diagnostics is performed also be modernised. With every plasma sample sent for diagnostics several separate tests are performed on small fractions of the sample to quantify proteins with known disease correlation instead of doing few tests analysing a larger number of targets. Multiplexed methods capable of analysing larger numbers of biomarkers in a single run have in recent years been developed. One of these methods is Immuno-SILAC that uses assets from the Human Protein Atlas, such as QprESTs and polyclonal antibodies to target and quantify proteins in plasma by mass spectrometric analysis with reduction of analysis time down to 15 minutes, which makes it a relevant method for clincs. To further increase the relevancy of Immuno-SILAC in clinics, evaluation of protocols essential for the function and therefore also limiting for the use of Immuno-SILAC, e.g. trypsin digestion, os of great importance. Aspects to take into evaluation within trypsin digestion are trypsin alternatives, time dependency and effects of denaturing agents such as urea. Improvements in these aspects could reduce the cost of sample preparation, time needed for digestion and increase the columes possible for analysis to increase the medical relevancy of Immuno-SILAC in clinics and make it a viable alternative method to current diagnostics.
During this master thesis, 7 known diagnostic targets were chosen and evaluated for the development of a PRM-assay, and absolute quantification in plasma with Immuno-SILAC. Aspects such as the effect of urea on trypsin, time dependency of digestion, and alternative trypsin enzymes were evaluated to increase the clinical applicability of Immuno-SILAC further.
Trypsin evaluation yielded positive results showing peptides with close-to unchaninging ratios over time. Trypsin showed close-to unchanged activity up tp 2 M urea which will decrease the dilution needed for digestion and enable robotics for automatic sample preparation, and digestion with alternative cheaper digestion showed promise for further use in the Immuno-SILAC protocol.