Granted Patent B2

US 12,228,576 · App. 18/068,047 · Granted Feb 18, 2025

Inventors: Bhavinkumar Patel (Rockford, IL); John Rogers (Rockford, IL)

Assignee: Pierce Biotechnology, Inc.

G01N33/57496C07K16/18G01N33/541G01N33/57488G01N33/6848G01N33/6863G01N33/6872G01N2440/14

Abstract

Claims (23)

1. A method for determining the ratio of phosphorylated to non- phosphorylated AKT-mTOR pathway proteins, comprising

a. treating a biological sample with one or more antibodies capable of immunoprecipitating a phosphorylated AKT-mTOR target protein, and separately treating the same biological sample with one or more antibodies capable of immunoprecipitating a non-phosphorylated AKT-mTOR target protein;

b. digesting the immunoprecipitated AKT-mTOR pathway proteins;

c. adding a first and a second detectably labelled internal standard peptide of known amount to the digested proteins, wherein the first internal standard peptide has the same amino acid sequence as a phosphorylated AKT-mTOR pathway peptide used to identify the phosphorylated protein, and the second internal standard peptide has the same amino acid sequence as the non-phosphorylated AKT- mTOR pathway peptide used to identify the non-phosphorylated protein;

d. assaying the digested proteins and internal standards via mass spectrometry to determine the presence and amount of phosphorylated and non-phosphorylated AKT-mTOR pathway peptides, wherein the AKT-mTOR pathway peptide is selected from the group consisting of SEQ ID NO: 5, 21, 44-46, 53, 59, 66, 74, 75, 80, 82, 84, 87, 103, 104, 108, 126, 131, 132, 143, 153, 156, 160, 166, 174, 196, 200, 205 and 212, and

e. determining the quantity of AKT-mTOR phosphorylated and non-phosphorylated pathway proteins in the sample, and determining the ratio of phosphorylated to non-phosphorylated target proteins.

2. The method of claim 1 , wherein the biological sample is from a human.

3. The method of claim 1 , wherein the antibody to detect phosphorylated AKT-mTOR pathway protein comprises an antibody that binds to phosphorylated AKT, phosphorylated IGF1R, phosphorylated IRS, phosphorylated IRS1, phosphorylated mTOR, phosphorylated P70S6K, phosphorylated GSK3a, phosphorylated GSK3b, phosphorylated TSC2, phosphorylated PRAS40, or phosphorylated PTEN.

4. The method of claim 1 , wherein the antibody to detect non-phosphorylated AKT-mTOR pathway protein comprises an antibody that binds to AKT, IGF1R, IRS, IRS1, mTOR, P70S6K, GSK3a, GSK3b, TSC2, PRAS40, or PTEN.

5. The method of claim 1 , wherein the peptide is modified with a detectable label.

6. The method of claim 1 , wherein the antibody is selected from the antibodies that bind to AKT1, AKT (pan), AKT2, phosphorylated AKT2 (pSer474), phosphorylated AKT (pSer473), phosphorylated IGF-1R (Tyr1161/Tyr1165/Tyr1166), phosphorylated IGF1 Receptor (IGF1R) pTyr1158+1162+1163, phosphorylated IGF1R pTyr1161, phosphorylated IGF-I Receptor β (Tyr1131), phosphorylated Insulin Receptor β (Tyr1146), IGF-I/Insulin Receptor β, INSR/Insulin Receptor, α-Insulin Receptor β subunit, INSR/Insulin Receptor alpha, phosphorylated Insulin Receptor (Y972), IRS1, phosphorylated IRS1 (pSer312), phosphorylated IRS1 (Ser307 mouse/Ser312 human), phosphorylated IRS-1 (pSer1101), mTOR, phosphorylated mTOR (pSer2448), S6K, S6K1, phosphorylated p70 S6 Kinase (pThr389/pThr412), phosphorylated p70 S6 Kinase (pThr389), phosphorylated p70 S6 Kinase (pThr421/pSer424), phosphorylated GSK-3α/β (pSer21/pSer9), GSK3α, phosphorylated GSK-3α (Ser21), GSK-3β, phosphorylated GSK-3β (pSer9), phosphorylated Tuberin/TSC2 (pSer939), TSC2, S6 ribosomal protein, phosphorylated S6 ribosomal protein (pSer235+236), PRAS40, phosphorylated PRAS40 (pThr246), phosphorylated PTEN (pSer380), or PTEN.

7. The method of claim 6 , wherein the antibody is capable of immunoprecipitating more than one AKT-mTOR pathway protein.

8. The method of claim 1 , wherein a first antibody is capable of immunoprecipitating a phosphorylated AKT-mTOR pathway protein, and a second antibody is capable of immunoprecipitating a non-phosphorylated version of the AKT-mTOR pathway protein precipitated by the first antibody.

9. The method of claim 1 , wherein step a) comprises treating the sample with a labelled antibody capable of binding to the pathway protein to provide a labelled antibody-protein conjugate; and binding the labelled antibody-protein conjugate with a capture agent capable of binding to the labelled antibody to isolate the target protein from the sample.

10. The method of claim 1 , wherein the quantity of an AKT-mTOR pathway protein is determined by adding an internal standard peptide of known amount to the digested protein prior to mass spectrometry, wherein the internal standard peptide has the same amino acid sequence as the AKT-mTOR pathway peptide, and is detectably labeled, and determining the quantity of an AKT-mTOR pathway peptide by comparison to the internal standard.

11. The method of claim 1 , wherein the quantity of an AKT-mTOR pathway protein is determined by a method comprising comparing an amount of an AKT-mTOR pathway peptide in the sample to the amount of the same AKT-mTOR pathway peptide in a control sample.

12. The method of claim 1 , wherein the quantity of an AKT-mTOR pathway protein is determined by a method comprising comparing an amount of the AKT-mTOR pathway peptide to an internal standard peptide of known amount, wherein both the peptide in the biological sample and the internal standard peptide are selected from the group consisting of SEQ ID NO: 5, 21, 44-46, 53, 59, 66, 74, 75, 80, 82, 84, 87, 103, 104, 108, 126, 131, 132, 143, 153, 156, 160, 166, 174, 196, 200, 205 and 212, wherein the standard peptide is detectably labeled.

13. The method of claim 1 , wherein the internal standard peptide is selected from the group consisting of SEQ ID NO: 5, 21, 44-46, 53, 59, 66, 74, 75, 80, 82, 84, 87, 103, 104, 108, 126, 131, 132, 143, 153, 156, 160, 166, 174, 196, 200, 205 and 212.

14. The method of claim 1 , wherein the digesting comprises a protease or chemical digest.

15. The method of claim 1 , wherein the digestion is single or sequential.

16. The method of claim 1 , further comprising desalting after digestion and prior to mass spectrometry.

17. The method of claim 1 , wherein the AKT-mTOR pathway protein is selected from RAC-alpha serine/threonine-protein kinase (AKT1), RAC-beta serine/threonine-protein kinase (AKT2), insulin receptor (INSR), insulin-like growth factor 1 receptor (IGF1R), insulin receptor substrate 1 (IRS1), tuberin (TSC2), serine/threonine-protein kinase mTOR (mTOR), glycogen synthase kinase-3 alpha (GSK3a), glycogen synthase kinase-3 beta (GSK3b), GSK3a/GSK3b, ribosomal protein S6 kinase beta-1 (p70S6K; RPS6KB1), 40S ribosomal protein S6 (RPS6), proline-rich AKT1 substrate 1 (PRAS40; AKT1S1), and phosphatidylinositol 3,4,5-triphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN).

18. The method of claim 1 , wherein a lower limit of quantification is from 0.05 to 0.75 fmol.

Assignments (2)

Continuity (3)

References Cited (31)