Immunoassays are the backbone of clinical laboratory quantification of serum proteins. Unfortunately for many patients, reported concentrations of proteins from immunoassays are misleading and can affect their care. Interference from heterophilic antibodies or autoantibodies as well as the hook effect and poor concordance between assay platforms can all potentially lead to erroneous results. We set out to determine if tandem mass spectrometry had the potential to solve many of the problems associated with quantification of low abundance serum proteins using thyroglobulin as a canonical example.

Methods

Thyroglobulin was digested using trypsin under three different digestion conditions. The most reliably quantifiable peptide across conditions was synthesized as an internal standard peptide for use in isotope-dilution tandem mass spectrometry by incorporating six deuterons into the side chain of one amino acid. The amount of thyroglobulin in the international standard for thyroglobulin (BCR-457) was determined using isotope dilution mass spectrometry. The three most reliably quantifiable peptides were used to raise polyclonal antibodies in rabbits. Those antibodies were used to enrich the thyroglobulin-specific peptides from trypsin digests of human serum along with a stable isotope labeled internal standard peptide that had been spiked into the digests. The resulting peptides were analyzed by tandem mass spectrometry. External human serum calibrators were analyzed in parallel.

Results

Our preliminary method had a limit of detection of 2.6 ng/mL (4 pmol/L) thyroglobulin in human serum. The intraassay and interassay imprecision was 13.7-22.5%. Method comparison with a traditional sandwich chemiluminescent immunoassay revealed good correlation (r2 = 0.81).

Conclusion

Trypsin digestion destroys endogenous interfering antibodies and direct detection of peptide with isotope dilution may facilitate better concordance between laboratories. In addition, single site peptide enrichment from digests using antibodies will not suffer from saturation effects. In summary, tandem mass spectrometry may have an important role in the quantification of serum proteins in the clinical laboratory in the future.