SRM/MRM Assay for the Tumor Necrosis Factor Receptor Superfamily Member 8 (CD30) Protein

• US 20200132694A1
• Filed: 01/11/2017
• Published: 04/30/2020
• Est. Priority Date: 07/11/2014
• Status: Abandoned Application

• Current Assignee: Expression Pathology, Inc. (NantWorks, LLC)
• Original Assignee: Expression Pathology, Inc. (NantWorks, LLC)
• Inventors: Krizman, David B., Hembrough, Todd, Liao, Wei-Li
• Application Number:
  US15/404,144
• Publication Number:
  US 20200132694A1
• Time in Patent Office: Days
• CPC Class Codes: G01N 2030/8831 involving peptides or proteins
• CPC Class Codes: G01N 2333/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95 NGF-receptor G01N2333/71, TNF-receptor G01N2333/7151
• CPC Class Codes: G01N 2496/00 Reference solutions for assays of biological material
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 2800/56 Staging of a disease; Further complications associated with the disease
• CPC Class Codes: G01N 2800/7028 Cancer
• CPC Class Codes: G01N 30/72 Mass spectrometers mass spectrometers per se H01J49/00
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the tumor necrosis factor receptor superfamily member 8 protein (CD30) that are particularly advantageous for quantifying the CD30 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue reagents and protocol and the CD30 protein is quantitated in the Liquid Tissue sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of a CD30 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

Claims

1. A method for measuring the level of the tumor necrosis factor receptor superfamily member 8 protein (CD30) in a biological sample of formalin-fixed tissue, comprising detecting and quantifying the amount of one or more modified or unmodified CD30 fragment peptides in a protein digest prepared from said biological sample using mass spectrometry;

and calculating the level of modified or unmodified CD30 protein in said sample; and

wherein said level is a relative level or an absolute level.

2. The method of claim 1, further comprising the step of fractionating said protein digest prior to detecting and/or quantifying the amount of one or more modified or unmodified CD30 fragment peptides.

3. The method of claim 2, wherein said fractionating step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography.

4. The method of claim 1, wherein said protein digest of said biological sample is prepared by the Liquid Tissue protocol.

5. The method of claim 1, wherein said protein digest comprises a protease digest.

6. The method of claim 5, wherein said protein digest comprises a trypsin digest.

7. The method of claim 1, wherein said mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry.

8. The method of claim 7, wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring (mSRM).

9. The method of claim 1, wherein the CD30 fragment peptide comprises an amino acid sequence as set forth as SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, and SEQ ID NO;

5.

12. The method of claim 1, wherein the tissue is paraffin embedded tissue.

13. The method of claim 1, wherein the tissue is obtained from a tumor.

17. The method of claim 1, wherein quantifying the CD30 fragment peptide comprises comparing an amount of one or more CD30 fragment peptides comprising an amino acid sequence of about 8 to about 45 amino acid residues of CD30 as shown in SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, and SEQ ID NO;

5 in one biological sample to the amount of the same CD30 fragment peptide in a different and separate biological sample.

18. The method of claim 1, wherein quantifying one or more CD30 fragment peptides comprises determining the amount of the each of the CD30 fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the CD30 fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence.

19. The method of claim 18, wherein the internal standard peptide is an isotopically labeled peptide.

20. The method of claim 19, wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.

21. The method of claim 1, wherein detecting and quantifying the amount of one or more modified or unmodified CD30 fragment peptides in the protein digest indicates the presence of modified or unmodified CD30 protein and an association with cancer in the subject.

22. The method of claim 21, further comprising correlating the results of said detecting and/Le and quantifying the amount of one or more modified or unmodified CD30 fragment peptides, or the level of said CD30 protein to the diagnostic stage/grade/status of the cancer.

23. The method of claim 22, wherein correlating the results of said detecting and quantifying the amount of one or more modified or unmodified CD30 fragment peptides, or the level of said CD30 protein to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer.

24. The method of claim 1, further comprising selecting for the subject from which said biological sample was obtained a treatment based on the presence, absence, or amount of one or more CD30 fragment peptides or the level of CD30 protein.

25. The method claim 1, further comprising administering to the patient from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon amount of one or more modified or unmodified CD30 fragment peptides or the level of CD30 protein.

26. The method of claim 25, wherein said therapeutic agent binds the CD30 protein and/or inhibits its biological activity.

27. The method of claim 26, wherein the therapeutic agent is selected to specifically target CD30-expressing cancer cells.

29. The method of claim 9, wherein the CD30 fragment peptide has the amino acid sequence as set forth as SEQ ID NO:

1.

10-11. -11. (canceled)

14-16. -16. (canceled)

28. (canceled)