Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry.
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.
Hoofnagle
Andrew N
AN
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA. ahoof@u.washington.edu
Becker
Jessica O
JO
Wener
Mark H
MH
Heinecke
Jay W
JW
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007118:Immunoassay; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013954:Thyroglobulin / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification
D010446:Peptide Fragments; D013954:Thyroglobulin
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