Interfacing HPLC with MALDI- and ESI-MS for Automated High Sensitivity Analysis of Proteins and Peptides

The research described in this thesis has an interdisciplinary approach dealing with chemical, biological, and technical issues. One objective was to interface miniaturized liquid chromatography (LC) to mass spectrometry, i.e. MALDI-TOF MS and ESI-MS, in an unattended mode for high sensitivity determination of proteins and peptides. The coupling between capillary LC and MALDI was accomplished by employing a piezoelectric flow-through microdispenser. The column effluent was nano fractionated onto a MALDI target plate with a negligible dispersion, thus transferring the high-resolution separation from the LC system onto the target plate as discrete spots. In conjunction, a simple sample preparation technique was developed that facilitated automated and unattended MALDI-TOF MS. A methodology for rapid analysis of phosphorylation sites utilizing capillary LC-ESI/MS/MS is presented. The combination of microcolumn chromatography and tandem mass spectrometry was employed for differentiation of the phosphorylation status of the tyrosine kinase ZAP-70. An automated comprehensive two-dimensional (2D) chromatography system was developed for the mapping of proteins below 30 kDa. In addition, a sample preparation step using restricted access materials (RAMs) was built in-line prior the first dimension, enabling selective sample enrichment. Biological samples, e.g. human hemofiltrate, were used to demonstrate the high resolving power of the 2D-LC system.