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Abstract Background: Thyroglobulin (Tg) is a 660 kDa homodimer synthesized by the follicular cells of the thyroid gland, acting as a substrate in the production of the hormones triiodothyronine (T3) and thyroxine (T4). Accurate measurement of Tg using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA) workflow combined with liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Given the general complexity of this workflow it has been predominantly implemented in laboratories experienced in LC-MS/MS protein analysis. Here we report the development of a SISCAPA™ workflow for accurate measurement of thyroglobulin on an Andrew+™ Pipetting Robot, enabling easier adoption of this workflow.
Methods: Thyroglobulin certified reference material (T-113) (Merck, UK) was used to create calibrators in surrogate serum (chicken serum, Merck, UK). In-house QC material prepared in pooled serum (BioIVT, UK) and surrogate serum, were used to evaluate method precision. External Quality Assurance (EQA) serum samples (NEQAS, UK) were analyzed using the newly developed method and the quantified results were compared to the All Laboratory Trimmed Mean (ALTM) of the scheme. The Waters™ Andrew+™ Pipetting Robot with OneLab™ Software was used to fully automate the workflow from denaturant/reduction reagent addition to the serum samples through to elution of the Tg FSP tryptic peptide off the magnetic beads, without the need for user intervention.
Briefly, 250µL serum samples were added to a 96-well plate, treated with denaturation and reduction buffer (Deoxycholate, TCEP, Trizma™ Base) and mixed for 40 minutes at 37°C. Internal standard and trypsin were added and mixed for 30 minutes at 37°C. The samples were quenched with a protease inhibitor cocktail and mixed for 10 minutes. Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes at room temperature. The plate was transferred to a magnetic array plate and the beads were left to pull down for 2 minutes. The samples were discarded, and the wells were washed with PBS/CHAPS wash buffer for 1 minute. The plate was transferred to a magnetic array plate and the beads were left to pull down, the wash buffer was discarded, and the wash step was repeated again. Tg was eluted from beads through the addition of 2% acetonitrile and 0.5% formic acid and mixed for 10 minutes at room temperature. The plate was transferred to a magnetic array plate and the beads were left to pull down and the eluate was transferred to a fresh 96-well plate. The samples were centrifuged for 5 minutes, followed by fitting of the autosampler magnetic plate. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters XSelect™ HSS T3, 2.5µm, 2.1 x 50 mm Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer with a run time of 2.6 minute.
Results: The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 0.1 ng/mL with S/N (PtP) >10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all < 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements.
Conclusions: A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.
For Research Use Only. Not for Use in Diagnostic Procedures. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
