Solid-support fluorescent derivatization of picomoles of protein at low concentration with FITC

A new method based on solid-support reaction is described to realize fluorescent derivatization of proteins at concentrations as low as 10(-8)M. A simple, low-cost homemade capillary C18 cartridge was fabricated as the solid-support reactor. Using bovine serum albumin (BSA) as a test protein, we demonstrated that the protein can be captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. The analysis by nano-HPLC with laser-induced fluorescence (LIF) detection was described. The effect of reaction conditions on the derivatization has been evaluated and discussed. The use of the solid-support reactor allows easy handling of as little as 8.5 pmol of BSA. A fraction from weak anion-exchange chromatography (WAX) of human liver extract was used as an illustrative example of application to real samples.