LC-MS/MS Analysis of Serum Thyroglobulin for Clinical Research

Thyroglobulin (Tg) is a 660 kDa, dimeric glycoprotein produced by the follicular cells of the thyroid and used entirely within the thyroid gland, acting as a substrate in the synthesis of the thyroid hormones thyroxine (T4) and triiodothyronine (T3).

In the laboratory, thyroglobulin measurement can be complicated by the presence of anti-thyroglobulin antibodies (ATAs, alternatively referred to as TgAb). The presence of these antibodies can result in falsely low (or rarely falsely high) levels of reported thyroglobulin in immunoassays.

Alternatively, it has been shown that using liquid chromatography with tandem mass spectrometry (LC-MS/MS) can help overcome some of the limitations of ATAs in immunoassays. Implementation of Stable Isotope Standards with Capture by Anti-Peptide Antibodies (SISCAPA) enables capture and quantification of tryptic peptides that act as a surrogate measure for the target protein. This provides significant improvements in method selectivity and analytical sensitivity compared to analysis of tryptic peptides from a simple sample digestion.

In addition, by reducing the protein to the peptide level with digestion, the impact of ATAs are negated, so a more accurate result can be obtained.
Although the use of SISCAPA in a surrogate peptide workflow can be deemed more accurate, it is based on the use of multiple reagents, steps and incubation times.

The aim of this presentation is demonstrate the performance of a simplified workflow for the offline automated sample preparation of thyroglobulin using the Andrew+ pipetting robot, followed by LC-MS/MS analysis using the ACQUITY UPLC™ I-Class and Xevo™ TQ Absolute for clinical research.

Presenter: Dominic Foley (Senior Scientist, Waters Corporation)

Dominic Foley is a Senior Scientist working at Waters Mass Spectrometry Headquarters in Wilmslow, UK. Having joined the company in September 2013, he has specialized in the development of LC-MS/MS clinical research applications, with a particular focus on steroid hormone analysis. In his role, he also collaborates with clinical customers in the development and implementation of LC-MS/MS methods in their laboratories.

Dominic has worked in separation sciences for 13 years, following his ambition of improving the quality of healthcare. He started his career with ICON Development Solutions, a CRO in the north of the UK, where he worked as a LC-MS/MS method development and validation scientist supporting DMPK studies in drug discovery. In 2012, he joined Thermo Fisher Scientific to work in their Chromatography R&D team as a Technical Support Specialist, working on the development of new chromatography and sample preparation products.

Dominic received his BSc (Hons) in Biochemistry from the University of Manchester in 2006. He has presented his work at conferences around the world and has several publications in both the Microchemical Journal and the Journal of Chromatography A.