US 12,228,576 B2
Detection and quantification of AKT-mTOR pathway proteins
Bhavinkumar Patel, Rockford, IL (US); and John Rogers, Rockford, IL (US)
Assigned to Pierce Biotechnology, Inc., Rockford, IL (US)
Filed by Pierce Biotechnology, Inc., Rockford, IL (US)
Filed on Dec. 19, 2022, as Appl. No. 18/068,047.
Application 18/068,047 is a division of application No. 16/081,377, granted, now 11,561,226, previously published as PCT/US2017/022062, filed on Mar. 13, 2017.
Claims priority of provisional application 62/308,051, filed on Mar. 14, 2016.
Prior Publication US 2023/0213529 A1, Jul. 6, 2023
Int. Cl. G01N 33/574 (2006.01); C07K 16/18 (2006.01); G01N 33/541 (2006.01); G01N 33/68 (2006.01)
CPC G01N 33/57496 (2013.01) [C07K 16/18 (2013.01); G01N 33/541 (2013.01); G01N 33/57488 (2013.01); G01N 33/6848 (2013.01); G01N 33/6863 (2013.01); G01N 33/6872 (2013.01); G01N 2440/14 (2013.01)] 18 Claims
 
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.