Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry

Abstract

Background: Quantification of serum tumor markers plays an important role in determining whether patients treated for cancer require further therapy. Whereas large-scale proteomic efforts aim to identify novel tumor markers to facilitate early detection, optimization of methods for quantifying known tumor markers offers another approach to improving management of malignancies. For example, immunoassays used in clinical practice to measure established tumor markers suffer from potential interference from endogenous immunoglobulins and imperfect concordance across platforms-problems that also plague many other immunoassays. To address these important limitations, this study used peptide immunoaffinity enrichment in concert with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify thyroglobulin, a well-characterized tumor marker.

Methods: We identified 3 peptides in tryptic digests of thyroglobulin that were detected at low concentrations by tandem mass spectrometry, raised polyclonal antibodies to those peptides, and used the antibodies to extract the 3 corresponding peptides from tryptic digests of human serum. We quantified each endogenous peptide using LC-MS/MS and multiple reaction monitoring with external calibrators.

Results: The detection limit for endogenous thyroglobulin in serum was 2.6 microg/L (4 pmol/L). Direct comparison with immunoassay revealed good correlation (r(2) = 0.81).

Conclusions: Immunoaffinity peptide enrichment-tandem mass spectrometry can detect tryptic peptides of thyroglobulin at picomolar concentrations while also digesting the endogenous immunoglobulins that can potentially interfere with traditional immunoassays. Our observations suggest a general analytical strategy for using immunoaffinity isolation together with tandem mass spectrometry to quantify tumor antigens and other low-abundance proteins in human serum.

Figure 1. Quantification of international certified reference material (BCR-457) by isotope dilution MS/MS

Triplicate dilutions of the certified reference material were digested with trypsin. The concentration of peptide 1 was determined in the tryptic digests from the ratio of endogenous to internal standard peptide. Each replicate dilution series is represented with different symbols. The curve representing the average of the data points was fit to the equation y = Axⁿ, which was used to determine the dilution necessary to achieve a response of 1.0. Results represent those observed in three independent experiments.

Figure 2. Chromatography and tandem mass spectrometry of immunoaffinity purified thyroglobulin peptides

Immunoaffinity purified peptides were first loaded onto a peptide-trapping column before being resolved with a C18 analytical column, ionized using micro-ESI, and analyzed using multiple reaction monitoring (MRM). Elution profiles of peptides purified from a digest of human serum are shown (left panels). Fragment ion spectra were collected in the linear ion trap of the mass spectrometer at the same time as MRM data. They are shown for each peptide (right panels). V* represents a stable isotope-labeled valine residue.

Figure 3. LC-MS/MS response of endogenous thyroglobulin peptides vs. concentration

Shown are the ion current peak areas (left panels) and responses (peak area of endogenous/peak area internal standard peptide, right panels) for each peptide detected in a dilution series of a human serum sample containing 319 μg/LTg. The internal standard peptide is chemically identical to Peptide 1 and differs only in mass.

Figure 4. Correlation of Tg quantification by immunoaffinity enrichment-LC-MS/MS and an immunoassay

Results using immunoaffinity purification of peptides from tryptic digests with external calibration were compared with a standard clinical immunoassay for Tg in serum (left). The Deming regression equation (Analyse-It; Leeds, England, UK) is shown with the Pearson correlation coefficient (r²). The residuals of each data point from the regression line are also shown (right) with the standard error of the residuals (S_y|x). The estimate of the true value was calculated using the ratio of the standard deviations of each method with Analyse-It.