Separations coupled to electrospray mass spectrometry

Proteins are mostly characterized using mass spectrometry (MS) after proteolysis. However, it should be difficult to analyze specific group of proteins ,e.g. phosphopeptides, by MS due to lower ionization efficiency in the presence of other peptides. Therefore, an efficient enrichment using selective affinity techniques prior to MS detection is necessary to increase their ionization efficiency and detection sensitivity from highly complex peptide mixtures. We have developed a new monolithic capillary column with an iron oxide nanoparticle coating for enrichment of phosphopeptides. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent electrostatic interactions of citrate ions on the surface of iron oxide nanoparticles with a quaternary amine functionalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. The affinity of iron oxide nanoparticles towards phosphorylated compounds was demonstrated by the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests, and their MALDI/MS characterization in off-line mode. The nanoparticle coating provides a significantly higher stability, capacity and selectivity, when compared with other monolithic materials. The developed column demonstrates its potential as a new tool for phosphoproteomic analysis. We have developed microfabricated electrospray microfluidic blocks based on the tip-less nebulizer design. The device shown in Image 1 is currently under testing and performs well at high nl/minute flow rates. It is anticipated, that even better performance could be achieved at decreased flow rates where the analyte suppression effects will be minimized.

The IACH group is equipped with the tools and basic chemicals necessary for the construction of the analytical microdevices including separation channels and detectors. The in house equipment including laser lithograph, spin coater, flow boxes and high-temperature furnace for heat treatment of a glass materials will form the basis for microfabrication. Two commercial automated systems: Capillary Electrophoresis BioFocus 3000 (BioRad) and liquid chromatograph LC10-AS (Shimadzu) both with scanning UV/VIS detectors, autosamplers and fraction collectors are available for routine analyses, methodology optimizations and sample micro preparations.

Project leader: 
František Foret
Image: Microfabricated electrospray microfluidic blocks based on the tip-less nebulizer design