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Title:
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Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads
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Author:
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Peró Gascón, Roger; Pont Villanueva, Laura; Benavente Moreno, Fernando J. (Julián); Barbosa Torralbo, José; Sanz Nebot, María Victoria
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Other authors:
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Universitat de Barcelona |
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Abstract:
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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. |
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Subject(s):
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-Electroforesi capil·lar
-Espectrometria de masses
-Camps magnètics
-Capillary electrophoresis
-Mass spectrometry
-Magnetic fields |
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Rights:
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(c) Wiley-VCH, 2016
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Document type:
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Article
Article - Accepted version |
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Published by:
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Wiley-VCH
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