title: Triple bioaffinity mass spectrometry concept for thyroid transporter ligands.
abstract: For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone (13)C(6)-L-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model "unknown" EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.
authors: RIKILT-Institute of Food Safety, Wageningen UR, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands.
authors: Payam
authors: P
authors: Aqai
authors: Christos
authors: C
authors: Fryganas
authors: Mineyuki
authors: M
authors: Mizuguchi
authors: Willem
authors: W
authors: Haasnoot
authors: Michel W F
authors: MW
authors: Nielen
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D052244:Endocrine Disruptors / Q000378:metabolism / Q000652:urine; D008024:Ligands; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013974:Thyroxine / Q000378:metabolism / Q000652:urine
chemical_list: D002247:Carbon Isotopes; D052244:Endocrine Disruptors; D008024:Ligands; D011228:Prealbumin; D011994:Recombinant Proteins; D013974:Thyroxine