Quantification of Thyroglobulin, a Low-Abundance Serum Protein, by Immunoaffinity Peptide Enrichment and Tandem Mass Spectrometry

Hoofnagle, Andrew N.; Becker, Jessica O.; Wener, Mark H.; Heinecke, Jay W.

doi:10.1373/clinchem.2008.109652

Cited by 284 publications

(241 citation statements)

References 32 publications

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“…Keshishian et al (10) showed that combining immuno-affinity depletion of proteins and limited chromatographic fractionation of peptides improves the LOD of SID-MRM-MS assays ϳ1000-fold (ϳ1 ng/ml) with intralaboratory CVs of 10 -25% when starting with 100 l of plasma. Here we have shown that the interlaboratory performance of multiplexed immuno-MRM-MS assays are consistent with those observed in prior studies conducted in single laboratories (15,16,18), as well as by MRM-MS and fMRM-MS. Importantly, by enriching for target peptides using anti-peptide antibodies prior to SID-MRM-MS, LOD improved ϳ1000-fold without increasing the interlaboratory imprecision relative to these other approaches.…”

Section: Discussionsupporting

confidence: 90%

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Kuhn

Whiteaker

Mani

et al. 2012

Molecular & Cellular Proteomics

168

158

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The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 l of plasma. Assay reproducibility was acceptable for verification studies, with median intra-and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-

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Section: Discussionsupporting

confidence: 90%

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Kuhn

Whiteaker

Mani

et al. 2012

Molecular & Cellular Proteomics

168

158

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“…Trypsin digestion and immunocapture of Tg-specific peptides followed by mass spectrometric analysis have been proposed as a possible solution for antibody interference, 6 but currently these methods do not have the required sensitivity for clinical applications and are relatively cumbersome compared with sandwich immunoassays.…”

Section: Discussionmentioning

confidence: 99%

Concordance between thyroglobulin antibody assays

Taylor¹,

Parkington²,

Bradbury³

et al. 2011

Ann Clin Biochem

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Background: Antithyroglobulin antibodies are a prevalent cause of interference in serum thyroglobulin immunoassays. Current guidelines recommend that antithyroglobulin antibodies should be measured concurrently with thyroglobulin when monitoring thyroid cancer patients post-thyroidectomy. However, the concordance between different antithyroglobulin assays has been questioned despite the availability of an international thyroglobulin antibody Reference Preparation. Methods: Four antithyroglobulin assays currently in use in UK laboratories (Siemens Immulite

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“…Measurement of Tg using peptide immunoaffinity enrichment with liquid chromatography-tandem mass spectrometry (Tg-LC/MS) has been proposed as a clinically accurate alternative method that is not influenced by the presence of anti-TgAbs (3)(4)(5)(6). Indeed, the Tg-LC/MS assay is offered at a number of clinical laboratories as a test to be performed reflexively as a measure of Tg instead of Tg-IMA in the presence of anti-TgAbs.…”

Section: Introductionmentioning

confidence: 99%

Thyroglobulin Liquid Chromatography–Tandem Mass Spectrometry Has a Low Sensitivity for Detecting Structural Disease in Patients with Antithyroglobulin Antibodies

Azmat

Porter

Senter

et al. 2017

Thyroid

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Background: Thyroglobulin (Tg) measurement in patients with positive antithyroglobulin antibodies (antiTgAbs) is not reliable. Tg measurement using liquid chromatography-tandem mass spectrometry (LC/MS) may be useful in this setting. Methods: This is a retrospective study with the objective of determining the accuracy of Tg-LC/MS in patients with thyroid cancer with anti-TgAbs. All patients with follicular cell-derived thyroid cancer (TC) who had thyroglobulin measured using LC/MS assay from November 1, 2013, to November 7, 2014, were evaluated. The frequency of detectable Tg-LC/MS was evaluated, with a functional sensitivity (FS) of 0.5 ng/mL in patients with structural disease. Then performance of Tg-LC/MS versus Tg immunometric assay (IMA) was compared using either Immulite assay (Tg-1) with a FS of 0.9 ng/mL or Beckman assay (Tg-B) with a FS of 0.1 ng/mL in detecting structural disease in patients with positive anti-TgAbs. Results: A total of 154 consecutive patients were included in this evaluation. Of these, 116 (75%) patients were positive for anti-TgAbs. In patients with structural disease and positive anti-TgAbs, Tg-LC/MS was undetectable in 43.7% of patients. Then the diagnostic accuracy for structural disease of Tg-LC/MS was compared with each Tg-IMA assay separately. In the 26 patients with positive anti-TgAbs where a Tg-I assay was used, the sensitivity and specificity for detecting structural disease were 33.3% and 88.2%, respectively, for the Tg-I assay, and 44.4% and 94.1%, respectively, for the Tg-LC/MS assay. In the 74 patients with positive anti-TgAbs where Tg-B was used, the sensitivity and specificity for detection of structural disease were 72.7% and 71.4%, respectively, for the Tg-B assay, and 62.6% and 93.7%, respectively, for the Tg-LC/MS assay. Conclusion: In patients with thyroid cancer with positive anti-TgAbs, Tg-LC/MS was frequently undetectable and was less sensitive for detecting disease than a Tg assay was with a functional sensitivity of 0.1 ng/mL. For patients with detectable Tg-LC/MS and anti-TgAbs, use of the assay for monitoring requires further prospective studies.

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Quantification of Thyroglobulin, a Low-Abundance Serum Protein, by Immunoaffinity Peptide Enrichment and Tandem Mass Spectrometry

Cited by 284 publications

References 32 publications

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Concordance between thyroglobulin antibody assays

Thyroglobulin Liquid Chromatography–Tandem Mass Spectrometry Has a Low Sensitivity for Detecting Structural Disease in Patients with Antithyroglobulin Antibodies

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