Quantification of Thyroglobulin, a Low-Abundance Serum Protein, by Immunoaffinity Peptide Enrichment and Tandem Mass Spectrometry
Open accessAbstract
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 μg/L (4 pmol/L). Direct comparison with immunoassay revealed good correlation (r2 = 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.
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Most known neosubstrate proteins have been identified through proteomic analyses of different cell lineages.
“Most known neosubstrate proteins have been identified through proteomic analyses of different cell lineages11,15,165,166.”
From Cancer therapies based on targeted protein degradation — lessons learned with lenalidomide (2021) · cites this paper as [166]
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Replacing Immunoassays With Tryptic Digestion-Peptide Immunoaffinity Enrichment And Lc–MS/MS
Becker, J. O., & Hoofnagle, A. N. (2012) · Bioanalysis · doi:10.4155/bio.11.319 · Cited by 97 · see citing works
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Liquid chromatography tandem mass spectrometry in the clinical laboratory
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Carvalho, V. M. (2012) · Journal of Chromatography B · doi:10.1016/j.jchromb.2011.08.027 · Cited by 90 · see citing works
A review of clinical diagnostic applications of liquid chromatography–tandem mass spectrometry
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“(23, 24); when tracking protein in a whole plasma hydrolysate,”
BackgroundBioanalysis of recombinant proteins and antibodies by mass spectrometry
Ezan, É., Dubois, M., & Bécher, F. (2009) · The Analyst · doi:10.1039/b819706g · Cited by 92 · see citing works
Hoofnagle, A. N., Wu, M., Gosmanova, A. K., Becker, J. O., Wijsman, E. M., Brunzell, J. D., Kahn, S. E., Knopp, R. H., Lyons, T. J., & Heinecke, J. W. (2010) · Arteriosclerosis Thrombosis and Vascular Biology · doi:10.1161/atvbaha.110.212894 · Cited by 87 · see citing works
Self-citationMass spectrometry of peptides and proteins from human blood
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Amacher, D. E. (2010) · Toxicology and Applied Pharmacology · doi:10.1016/j.taap.2010.02.011 · Cited by 85 · see citing works
Targeted Quantification of Low ng/mL Level Proteins in Human Serum without Immunoaffinity Depletion
Shi, T., Sun, X., Gao, Y., Fillmore, T., Schepmoes, A., Zhao, R., He, J., Moore, R., Kagan, J., Rodland, K., et al. (2013) · Journal of Proteome Research · doi:10.1021/pr400178v · Cited by 79 · see citing works
Thyroglobulin measurement by highly sensitive assays: focus on laboratory challenges
Giovanella, L., Feldt‐Rasmussen, U., Verburg, F. A., Grebe, S. K. G., Plebani, M., & CLARK, P. M. S. (2014) · Clinical Chemistry and Laboratory Medicine (CCLM) · doi:10.1515/cclm-2014-0813 · Cited by 83 · see citing works
Global Stability of Plasma Proteomes for Mass Spectrometry-Based Analyses
Zimmerman, L. J., Li, M., Yarbrough, W. G., Slebos, R. J. C., & Liebler, D. C. (2012) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m111.014340 · Cited by 83 · see citing works
“We then compared the LC-MS/MS data sets to detect differences in (1) the numbers of MS/MS spectra collected, (2) confident peptide identifications (numbers of MS/MS spectra matched to peptides), (3) unique peptide identifications, (4) protein group identifications (numbers of indistinguishable proteins as defined in (…”
de Jong, W. H. A., de Vries, E. G. E., & Kema, I. P. (2010) · Clinical Biochemistry · doi:10.1016/j.clinbiochem.2010.07.006 · Cited by 80 · see citing works
Diffusing protein binders to intrinsically disordered proteins
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“21.1_5286) 45 .”
InfluentialSelf-citationControversies in the Management of Low-Risk Differentiated Thyroid Cancer
Haymart, M. R., Esfandiari, N. H., Stang, M. T., & Sosa, J. A. (2017) · Endocrine Reviews · doi:10.1210/er.2017-00067 · Cited by 79 · see citing works
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The evolving role of mass spectrometry in cancer biomarker discovery
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“This strategy has been reported for thyroglobulin, a protein with known autoantibody interferences [9,10].”
BackgroundPrakash, A., Rezai, T., Krastins, B., Sarracino, D. A., Athanas, M., Russo, P. S., Ross, M. M., Zhang, H., Tian, Y., Kulasingam, V., et al. (2010) · Journal of Proteome Research · doi:10.1021/pr100821m · Cited by 74 · see citing works
The application of quantification techniques in proteomics for biomedical research
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“The method of stable isotope standards and capture by anti-peptide antibodies (SISCAPA) was developed by Anderson et al. using peptide-based antibodies to enrich targeted low abundance peptides (Anderson et al., 2004; Hoofnagle et al., 2008).”
MethodGerszten, R. E., Asnani, A. H., & Carr, S. A. (2011) · Circulation Research · doi:10.1161/circresaha.110.225003 · Cited by 69 · see citing works
Ocaña, M. F., & Neubert, H. (2010) · Analytical Biochemistry · doi:10.1016/j.ab.2010.01.002 · Cited by 69 · see citing works
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Ringel, M. D., & Nabhan, F. A. (2013) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/jc.2013-1412 · Cited by 67 · see citing works
Paterson, R., Heywood, W., Heslegrave, A., Magdalinou, N., Andreasson, U., Sirka, E., Bliss, E., Slattery, C., Toombs, J., Svensson, J., et al. (2016) · Translational Psychiatry · doi:10.1038/tp.2016.194 · Cited by 65 · see citing works
Clinical applications of mass spectrometry‐based proteomics in cancer: Where are we?
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Thyroglobulin measurement in the management of patients with differentiated thyroid cancer
Algeciras-Schimnich, A. (2018) · Critical Reviews in Clinical Laboratory Sciences · doi:10.1080/10408363.2018.1450830 · Cited by 63 · see citing works
“[47] and other methods have been published subsequently [37,48,49].”
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Xu, Y., Mehl, J. T., Bakhtiar, R., & Woolf, E. (2010) · Analytical Chemistry · doi:10.1021/ac1009832 · Cited by 64 · see citing works
Application of Proteomics in Biomarker Discovery: a Primer for the Clinician
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Quantification of growth hormone in serum by isotope dilution mass spectrometry
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Lee, A. Y. H., Chappell, D. L., Bak, M. J., Judo, M., Liang, L. A., Churakova, T., Ayanoglu, G., Castro‐Perez, J. M., Zhou, H., Previs, S. F., et al. (2015) · Clinical Chemistry · doi:10.1373/clinchem.2015.244251 · Cited by 59 · see citing works
Advances in Proteomic Technologies and Its Contribution to the Field of Cancer
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“, who implemented SISCAPA assays for quantification of low-abundance serum thyroglobulin, and simultaneous measurement of apolipoprotein A–I and apolipoprotein B [133, 173].”
BackgroundMethodFirst Steps toward Harmonization of LC-MS/MS Thyroglobulin Assays
Netzel, B. C., Grant, R. P., Hoofnagle, A. N., Rockwood, A. L., Shuford, C. M., & Grebe, S. K. G. (2015) · Clinical Chemistry · doi:10.1373/clinchem.2015.245266 · Cited by 58 · see citing works
Self-citationSchoenherr, R. M., Zhao, L., Whiteaker, J. R., Feng, L.-C., Li, L., Liu, L., Liu, X., & Paulovich, A. G. (2009) · Journal of Immunological Methods · doi:10.1016/j.jim.2009.11.017 · Cited by 57 · see citing works
“) These performance characteristics are consistent with those reported in polyclonal antibody-based SISCAPA assays,(Berna et al., 2006; Whiteaker et al., 2007a; Hoofnagle et al., 2008; Kuhn et al., 2009) demonstrate the feasibility of using a monoclonal antibody for SISCAPA, and raise the possibility of identifying ve…”
MethodResultAzmat, U., Porter, K. M., Senter, L. A., Ringel, M. D., & Nabhan, F. A. (2016) · Thyroid · doi:10.1089/thy.2016.0210 · Cited by 57 · see citing works
Jung, S., Whiteaker, J. R., Zhao, L., Yoo, H., Paulovich, A. G., & Hahn, S. H. (2016) · Journal of Proteome Research · doi:10.1021/acs.jproteome.6b00828 · Cited by 55 · see citing works
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Emerging role of clinical mass spectrometry in pathology
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Biochemical Testing in Thyroid Disorders
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Multiple Reaction Monitoring (MRM)
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Rappold, B. A. (2021) · Annals of Laboratory Medicine · doi:10.3343/alm.2022.42.2.121 · Cited by 52 · see citing works
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Mass spectrometry-based targeted proteomics for analysis of protein mutations
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Schoenherr, R. M., Whiteaker, J. R., Zhao, L., Ivey, R. G., Trute, M., Kennedy, J. J., Voytovich, U. J., Yan, P., Lin, C., & Paulovich, A. G. (2012) · PROTEOMICS · doi:10.1002/pmic.201100587 · Cited by 50 · see citing works
“[27] propose that the peptides hydrolyze slowly from the resin.”
BackgroundKaiser, P., Akerboom, T. P. M., Ohlendorf, R., & Reinauer, H. (2010) · Clinical Chemistry · doi:10.1373/clinchem.2009.139477 · Cited by 50 · see citing works
The current status of clinical proteomics and the use of MRM and MRM3for biomarker validation
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Boja, E. S., & Rodriguez, H. (2011) · Annals of Laboratory Medicine · doi:10.3343/kjlm.2011.31.2.61 · Cited by 49 · see citing works
“LDTs have historically been a general subject of FDA enforcement discretion.”
MethodInfluentialKennedy, J. J., Whiteaker, J. R., Schoenherr, R. M., Yan, P., Allison, K. H., Shipley, M., Lerch, M. L., Hoofnagle, A. N., Baird, G. S., & Paulovich, A. G. (2016) · Journal of Proteome Research · doi:10.1021/acs.jproteome.6b00245 · Cited by 48 · see citing works
Self-citation
- Authors
- Hoofnagle, A. N., Becker, J. O., Wener, M. H., & Heinecke, J. W.
- Affiliations
- Departments of Laboratory Medicine and
- University of Washington (US)
- Corresponding author
- Hoofnagle, Andrew N
- Venue
- Clinical Chemistry
- Publisher
- Oxford University Press (OUP)
- Published
- 2008-11-01
- Volume
- 54
- Issue
- 11
- Pages
- 1796-1804
- ISSN
- 0009-9147 (ISSN-L)
- 1530-8561
- PMC
- PMC2739673
- OpenAlex
- W2167033601
- Cited by
- 314
- References
- 34
- Citations by year
- 2026: 2 · 2025: 9 · 2024: 12 · 2023: 13 · 2022: 9 · 2021: 10 · 2020: 16 · 2019: 9 · 2018: 16 · 2017: 21 · 2016: 25 · 2015: 15 · 2014: 28 · 2013: 26 · 2012: 31
- FWCI
- 10.09
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Clinical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- University of Washington
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- thyroglobulin, tumor markers, immunoassay interference, polyclonal antibodies, tryptic peptides, multiple reaction monitoring, detection limit
- MeSH terms
- Amino Acid Sequence, Chromatography, Affinity, Chromatography, High Pressure Liquid, Humans, Immunoassay, Peptide Fragments, Peptide Mapping, Reference Standards, Sensitivity and Specificity, Thyroglobulin, Reproducibility of Results, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry
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