OR14-2 A 2nd generation mass spectrometric serum thyroglobulin (Tg) assay achieves analytical performance similar to many immunometric Tg assays and is immune to Tg autoantibody interferences

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR14-Thyroid Cancer: Insights into Diagnosis & Treatment
Clinical
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 11:45 AM
Room 103 (Moscone Center)
Brian C Netzel*1, Robert L Taylor1, Ravinder Jit Singh1 and Stefan Karl Gunther Grebe2
1Mayo Clinic, Rochester, MN, 2Mayo Clinic and Foundation, Rochester, MN
Introduction: Serum thyroglobulin (Tg) testing is a key part of thyroid cancer follow-up. However, Tg auto-antibodies (TgAB) can lead to false-low/false-negative measurements in standard immunometric assay. Recently, it has been shown that proteomic Tg analysis by mass spectrometry (MS) overcomes TgAB interferences. Tg, and any TgAB, in patient serum are digested using proteases. Tg-proteotypic peptides can then be monitored and quantified to give true Tg concentrations regardless of TgAB status. However, 1st generation Tg MS assays showed suboptimal detection sensitivity. To improve this careful consideration must be given to peptide selection and specimen preparation, as these will affect assay sensitivity and specificity.

Materials and Methods: 250mL of serum was selectively depleted of middle and low molecular weight proteins. After re-suspension, specimens were reduced, alkylated and digested with trypsin for 16h. Following trypsin inactivation, we used protein-G paramagnetic beads (Life Technologies, Carlsbad, CA) with bound anti-peptide monoclonal antibodies against the peptide sequence FSPDDSAGASALLR (SISCAPA Assay Technologies, Vancouver, BC) for further purification, before analyis on an API 5000 triple quadrupole mass spectromter (AB SCIEX, Foster City, CA). MS analysis was performed in positive electrospray ionization (ESI) mode, monitoring three transitions for both the proteotypic peptide and its internal standard.

Results: Compared with a 1st generation Tg MS assay, which we have previously described (1), the choice of a different peptide target combined with target peptide enrichment reduced the limits of detection (LOD) and quantification (LOQ) 5-10 fold,  from 2 and 5ng/mL to 0.2 and 1ng/mL, respectively. Method agreement between the two generations of MS assays (N = 48) was excellent (slope: 1.01, y-intercept: 0.40, R2: 0.991). Comparison with the Beckman DXI immunoassay (N = 70) in TgAB-negative patients showed a slope of 0.95, intercept 1.2 and R2 0.91. In TgAb positive specimens (N= 42) the corresponding figures were slope: 1.44, y-intercept: 0.02, and R2: 0.97.  Imprecision (CV) was 4.1 – 7.1% (Tg range 2 – 100ng/mL). Spiked recovery (% predicted) across the detectable range was 87-110%.

Conclusion: Peptide selection and specimen preparation are crucial for optimal analytical sensitivity and specificity of Tg by MS. Factors to be considered include ionization potential of individual peptides, ease of specific fragmentation, and absence of peptide polymorphisms in the target peptide that might render it invisible to MS detection. Based on these principles, our 2nd generation Tg MS assay now achieves a LOD and LOQ that make it suitable as a viable alternative for modern immunometric assays, when TgAB interference is suspected.

(1) Taylor, RL et al., Endocrine Abstracts (2012) 29:1784

Nothing to Disclose: BCN, RLT, RJS, SKGG

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