Comparison of capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry for the analysis of transthyretin in human serum

摘要

Capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-MS and CapLC-MS, respectively) are nowadays very suitable techniques for the separation and characterization of intact proteins in biological fluids. In this paper, we compare the performance of both techniques for the analysis of transthyretin (TTR), which is a homotetrameric protein (relative molecular mass (M-r) similar to 56,000) involved in different types of amyloidosis. Furthermore, it is also presented a novel sample pretreatment based on immunoprecipitation (IP) using Protein A Ultrarapid Agarose (TM) (UAPA) magnetic beads (MBs) to purify TTR from serum samples. This novel IP based on MBs allowed the detection of TTR monomeric proteoforms that were not possible to analyze by conventional IP in solution. In addition, UAPA MBs provided many other desirable advantages including higher selectivity and minimal unspecific binding of other proteins. CE-MS and CapLC-MS were applied to analyze serum samples from healthy controls and familial amyloidotic polyneuropathy type I (FAP-I) patients, who suffered from the most common hereditary systemic amyloidosis. Both techniques allowed detecting the same TTR proteoforms, including the mutant TTR (Met 30) variant (variation in relative molecular mass (Delta M-r) was +32.07, from wild-type TTR). Migration/retention times and relative quantitation of the different proteoforms were similar and reproducible in both cases, but the limits of detection (LOD5) achieved by CE-MS were slightly lower (2-2.5-fold). Some other differences were also found on separation selectivity (migration orders and separation of antibody), peak efficiency, total analysis time, calibration ranges and experimental M-r accuracy. (C) 2016 Elsevier B.V. All rights reserved.