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oai:recercat.cat:2445/1231872025-12-04T19:37:09Zcom_2072_1057col_2072_478813col_2072_478917

Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads Peró Gascón, Roger Pont Villanueva, Laura Benavente Moreno, Fernando J. (Julián) Barbosa Torralbo, José Sanz Nebot, María Victoria Electroforesi capil·lar Espectrometria de masses Camps magnètics Capillary electrophoresis Mass spectrometry Magnetic fields In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid AgaroseTM (UAPA), Dynabeads® Protein A (DyPA) and SiMAG-Protein A (SiPA)) and AffiAmino Ultrarapid AgaroseTM (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases results were repeatable and it was possible the identification and the quantitation of the relative abundance of the 6 most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimised conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3 % RSD for migration times and peak areas) were good, the method was linear between 5- 25 µg·mL-1 and limit of detection (LOD) was around 1 µg·mL-1 (25 times lower than by CE-MS, 25 µg·mL-1). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients. 2018-06-21T14:04:35Z 2018-06-21T14:04:35Z 2016 2018-06-21T14:04:36Z info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion Versió postprint del document publicat a: https://doi.org/10.1002/elps.201500495 Electrophoresis, 2016, vol. 37, p. 1220-1231 https://doi.org/10.1002/elps.201500495 (c) Wiley-VCH, 2016 info:eu-repo/semantics/openAccess Wiley-VCH Articles publicats en revistes (Enginyeria Química i Química Analítica)