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Thyroglobulin quantitation by mass spectroscopy

A technology of thyroglobulin and mass spectrometry, applied in the field of quantitative thyroglobulin, which can solve the problems of interference in clinical application, interference, hook effect, etc.

Pending Publication Date: 2015-07-22
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all face methodological issues, such as differences in standardization, variability in assay sensitivity and precision between batches, hook effects, and interference by Tg antibodies
The interference problem caused by Tg antibodies especially makes it difficult to monitor the clinical application of Tg levels as a tumor marker because up to 20% of thyroid cancer patients have Tg autoantibodies

Method used

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  • Thyroglobulin quantitation by mass spectroscopy
  • Thyroglobulin quantitation by mass spectroscopy
  • Thyroglobulin quantitation by mass spectroscopy

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0069] Filtration is a preferred method of preparing samples for chromatography, especially biological samples such as serum or plasma. This filtration is performed by filtering the sample through a molecular weight cut-off filter to separate species having molecular weights above the filter cut-off (including the Tg) from species having molecular weights below the filter cut-off. The sample remaining above the filter after complete (or near complete) filtration is substantially free of potentially interfering substances with molecular weights below the filter cut-off.

[0070] The pH of the sample can then be adjusted to any point desired for the digest. In certain preferred embodiments, the digestive agent is trypsin and the pH can be adjusted using ammonium acetate solution to make the pH suitable for the enzyme. In these preferred embodiments, the sample is then trypsinized to form Tg peptides (including peptide T129).

[0071] Following trypsinization, the sample can be...

Embodiment 1

[0100] Example 1 : Illustration of MS quantification of peptide T129

[0101]Several samples with various known concentrations of peptide T129 were prepared by serial dilution starting from a sample of known peptide T129 concentration. The LOQ and calibration curve for peptide T129 were generated from LC-MS / MS analysis of these samples.

[0102] Utilize Phenomenex analysis column (Phenomenex Corp. Luna 5 μ C8 (2) New Column 50x1.0mm) for LC. A binary HPLC eluent consisting of 0.2% formic acid in ultrapure water (HPLC grade) (mobile phase A) and 0.2% formic acid in 100% methanol (mobile phase B) was applied to the analytical column to extract from the sample containing Separate selected Tg peptides from other substances. The binary eluent was applied according to the following gradient format: first step, an 80 / 20 mobile phase A / mobile phase B mixture was applied for 120 seconds; second step, a 30 / 70 mobile phase A / mobile phase B mixture was applied for 60 seconds ; In...

Embodiment 2

[0112] Example 2: Illustration of quantification of peptide T129 in spiked, concentrated and digested serum

[0113] On top of the filter element of a commercially available 300 kDa molecular weight cut-off filter cartridge (Pall Corp. Nanosep 300 kDa, Pall Corp. Cat. No. OD300C33) was added 500 μl of serum-depleted sample (e.g., the sample in this example ).

[0114] After centrifuging the core at 13 kg for 6 minutes, the sample was completely filtered. Remove and discard the filtrate. Then, 500 μl of HPLC grade water was added to the top of the filter and the core was centrifuged again at 13 kg for 6 minutes. The filtrate was then removed and discarded. Next, 200 μl of 20 mM ammonium acetate was added to the top of the filter. The core was again centrifuged at 13 kg for 3 minutes. Then, the filtrate was removed and discarded, and 100 μl of 20 mM ammonium acetate was added to the top of the filter.

[0115] Subsequently, 15 μg of trypsin (Promega Trypsin Gold, mass sp...

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Abstract

Provided are methods for determining the amount of thyroglobulin in a sample using various purification steps followed by mass spectrometry. The methods generally involve purifying thyroglobulin in a test sample, digesting thyroglobulin to form peptide T129, purifying peptide T129, ionizing peptide T129, detecting the amount of peptide T129 ion generated, and relating the amount of peptide T129 ion to the amount of thyroglobulin originally present in the sample.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application Serial No. 61 / 703,721, filed September 20, 2012, under subsection five of section 119 of title 35, United States Code (35 U.S.C. § 119(e)), which The entire contents are incorporated herein by reference. field of invention [0003] The present invention relates to the quantification of thyroglobulin. In a particular aspect, the invention relates to methods of quantifying thyroglobulin by mass spectrometry. Background of the invention [0004] The following description of the background to the disclosure is provided only as an aid in understanding the invention and is not considered to describe or constitute prior art to the present invention. [0005] Thyroglobulin or Tg is a large dimeric secreted glycoprotein with a molecular weight of 660 kDa consisting of non-covalently associated homodimers. [0006] Tg molecules exist in several forms. The th...

Claims

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Application Information

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IPC IPC(8): C12Q1/37
CPCG01N2800/046G01N33/78G01N33/6893G01N33/6848C12Q1/37G01N2333/47G01N2496/00G01N2560/00G01N2800/7028
Inventor Y·张N·J·克拉克R·E·赖茨
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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