Coupling of capillary-channeled polymer (C-CP) fibers for reversed phase liquid chromatography and ESI-MS for the determination of proteins in a urine matrix

Abstract

Electrospray ionization mass spectrometry (ESI-MS) analysis provides a great deal of analytical information as a detection mode when coupled with liquid chromatography (LC) and capillary electrophoresis (CE) separations of proteins. However, limitations to low flow rates (<0.5 mL min⁻¹) for optimal spectral clarity and greatest sensitivity for conventional ESI generally imply low linear velocities (∼4 mm s⁻¹ for packed bed formats) and slow LC separations. Capillary-channeled polymer (C-CP) fibers have been employed for fast protein separations, explicitly for the capability to operate at high linear velocities, without van Deemter C-term penalties. In order to maintain the preferred high linear velocities (>50 mm s⁻¹), while also using volume flow rates compatible with standard ESI-MS, smaller column diameters are evaluated with the intension to match the high chromatographic throughput demonstrated through UV-Vis absorbance detection with the high information fidelity of the MS detection. Mixtures of ribonuclease A, cytochrome c, myoglobin and lysozyme were prepared in phosphate buffered saline (PBS) and urine matrices. The efficiency of the matrix removal is reflected in the near-identical qualitative and quantitative responses in both cases. It is felt that C-CP microbore columns can provide the high throughput and solute recoveries desired in top-down proteomics applications.