On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry for the analysis of large...

The analysis of large biomolecules by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) remains unexplored because of the complex issues that need to be addressed. In this preliminary study, we used the human glycoprotein transferrin (Tf) as a model of a large biomolecule. First, we established by CE-UV a novel method compatible with IA-SPE-CE-MS, based on the use of a fused silica capillary coated with an anionic derivative of polyacrylamide (UltraTrol(TM) Dynamic Pre-Coat High Normal, HN) to prevent protein adsorption. The methodology allowed the detection of the most abundant Tf sialoforms. Repeatability studies demonstrated high stability of the coated capillaries, which was required for on-line immunoextraction and MS detection. IA-SPE-CE-UV and IA-SPE-CE-MS methods were optimized for the analysis of Tf standards and human serum samples using a laboratory-made IA sorbent. Three peaks corresponding to Tf were detected with UV detection when on-line immunoextraction was applied to the standards. The use of MS detection, however, reduced the resolution of the electrophoretic separation. Finally, we demonstrated that it was possible to detect Tf in human serum samples, after off-line serum sample de-salting by centrifugal filtration.