Patent ID: 8604692

Claim:
A method for simultaneously determining the levels of and/or phosphorylation states of a set of target proteins or peptides in a sample, said target proteins or peptides are eIF4E (NP — 001959.1), HuR (NP — 001410.2), and at least one eIF4E regulon component selected from the group consisting of Cyclin D1 (NP — 444284.1); NBS/Nibrin (NP — 002476.2); Pim-1 (NP — 002639.1); Cyclin B1 (NP — 114172.1); Cyclin A2 (NP — 001228.1); ODC (NP — 002530.1); VEGF (NP — 003367.4); Skp2 (NP — 005974.2, NP — 116026.1); Cyclin E1 (NP — 001229.1); c-myc (NP — 002458.2); FGF2 (NP — 001997.5); MMP-9 (NP — 004985.2); mdm2 (NP — 002383.2); caspase-9 (NP — 001220.2, NP — 127463.1); bc12 (NP — 000624.2, NP — 000648.2); Bcl/xL (NP — 612815.1); Selenoprotein S (NP — 060915.2); eIF4E-BP1 (NP — 004086.1); Akt1 (NP — 001014431.1, NP — 005154.2, NP — 001014432.1); PI3K (NP — 006209.2, NP — 002640.2); GSK3B (NP — 002084.2); and mTOR/FRAP1 (NP — 004949.1), the method comprising: (a) adding at least one internal standard protein or peptide corresponding to each target protein of eIF4E, HuR, and the at least one eIF4E regulon component selected from the group consisting of Cyclin D1 , NBS/Nibrin, Pim-1, Cyclin B1, Cyclin A2, ODC, VEGF, Skp2, Cyclin E1 , c-myc, FGF2, MMP-9, mdm2, caspase-9, bc12, Bc1/xL, Selenoprotein S, eIF4E-BP1, Akt1, PI3K, GSK3B, and mTOR/FRAP1to the sample; (b) fragmenting the target proteins or peptides of eIF4E, HuR, and the at least one eIF4E regulon component and the at least three corresponding internal standard proteins or peptides, thereby generating fragments of the eIF4E, the HuR, and the at least one eIF4E regulon component, and the at least three corresponding internal standard proteins or peptides; (c) analyzing the corresponding parent masses and determining amounts of the resultant fragments from step (b) by a mass spectrometry-based method ; and (d) using the results from step (c) to determine and quantitate the levels of and/or phosphorylation states of the eIF4E, HuR, and the at least one eIF4E regulon component.