Journal
TALANTA
Volume 170, Issue -, Pages 224-232Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2017.03.104
Keywords
Capillary electrophoresis; Fab' fragments; Mass spectrometry; On-line immunopurification; Transthyretin
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Funding
- Spanish Ministry of Economy and Competitiveness [BES-2012-061127]
- Spanish Ministry of Education and Science [CTQ2014-56777-R]
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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 (M-r similar to 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 25 mu g mL(-1), limits of detection (LODs) were around 0.5 mu g mL(-1) (50-fold lower than by CE-MS, similar to 25 mu g mL(-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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