Development of multidimensional liquid chromatographic methods hyphenated to mass spectrometry, preparation and analysis of complex biological samples

摘要

Immunoadsorbers based on monolithic epoxy-activated CIM disks have been developed in order to target biomarkers of heart diseases. The developed immunoadsorbers permitted to selectively isolate myoglobin and NT-proBNP from human serum. Anti-NT-proBNP-CIM disks permitted a quantitative isolation of NT-proBNP at concentrations down to 750 amol/µL in serum (R2 = 0.998).Six different restricted access materials have been evaluated with respect to their ability to remove hemoglobin from hemolysates. Experiments at different pH revealed that the retention of hemoglobin can be drastically diminished at pH 10.7. Because of better chemical stability at high pH, the polymeric Biotrap 500 MS RAM column was optimized for the analysis of hemolysates. The setup permits to quantitatively extract antibiotics from whole blood hemolysates at biologically relevant concentrations (200 pg/µL), and without carry-over of hemoglobin.A new 2D-HPLC-ESI-MS/MS setup for proteome analysis was developed. It consisted of a peptide separation by RP-HPLC at pH 10.0, followed by IP-RP-HPLC at pH 2.1. This new setup was compared with a classical SCX x IP-RP-HPLC setup. Separation repeatability is similar with both setups. The orthogonality between methods of separation is higher in the SCX x IP-RP-HPLC approach than in the RP x IP-RP-HPLC scheme. However, the better peptide distribution and separation efficiency achieved with the RP x IP-RP-HPLC setup permitted to identify significantly more peptides than with the classical SCX x IP-RP-HPLC setup. Both approaches are complementary and a combination of both setups permits to identify more peptides than replicate injections performed with a single setup.