On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin

Publication date: 1 August 2017
Source:Talanta, Volume 170
Author(s): Laura Pont, Fernando Benavente, José Barbosa, Victoria Sanz-Nebot
This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab′ antibody fragments (Fab′-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (Mr~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab′ fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab′-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL⁻¹, limits of detection (LODs) were around 0.5µgmL⁻¹ (50-fold lower than by CE-MS, ~25µgmL⁻¹) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab′-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability.

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