To purify and characterize low-abundance proteins in complex biological mixtures, we used a novel strategy that combined preparative two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative 2D-LPE is based on the same isoelectric focusing and gel electrophoresis principles as the widely used analytical 2D gel electrophoresis, except that analytes remain in solution throughout separation. This novel approach shows many improvements compared to analytical 2D gel electrophoresis for the separation of proteins in biological fluids. For example, larger volumes/amounts of samples can be loaded, yielding sufficient amounts of low-abundance proteins for further characterization. Since proteins remain in liquid phase during the entire procedure, extra steps such as electroelution, extraction, or transfer to membranes from the gels prior to mass spectrometric analysis are obviated. We report the usefulness of 2D-LPE combined with MALDI-TOF MS for the purification and characterization of cystatin C and β-2 microglobulin in human cerebrospinal fluid. This method should be applicable to a wide range of biological fluids, such as cerebrospinal fluid, serum, tissue extracts, cell media, whole cells, and bacterial lysates.
ASJC Scopus subject areas
- Analytical Chemistry