MRM-MS SIGNATURE ASSAY
The present invention relates to mass spectrometry methods employing multiple reaction monitoring (MRM) in the field of cancer therapeutics, specifically prostate cancer and melanoma.
1 . A method of determining the concentration of a prostate cancer biomarker or affiliated molecular partner in a sample comprising:
(a) obtaining a sample from a subject;
(b) treating said sample to digest one or more prostate cancer biomarkers or affiliated molecular partners contained therein;
(c) generating a mass spectrometry profile of the digested sample of step (b);
(d) comparing the mass spectrometry profile from step (c) to a standard curve, wherein said standard curve has been created using at least one calibration standard selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO 28, SEQ ID NO. 29 SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 and combinations thereof; and
(e) calculating a concentration of said one or more prostate cancer biomarkers or affiliated molecular partners in the sample obtained from the subject based on the standard curve.
2 . The method of claim 1 , wherein the prostate cancer biomarker is FASN.
3 . The method of claim 1 , wherein the affiliated molecular partner is USP2a.
4 . The method of claim 2 , wherein the affiliated molecular partner is USP2a.
5 . The method of claim 2 wherein the sample of step (b) is subjected to liquid chromatography prior to the generation of said mass spectrometry profile.
6 . The method of claim 1 , further comprising spiking the sample of (a) with a known concentration of one or more peptides or proteins selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO 28, SEQ ID NO. 29 SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 and combinations thereof.
7 . The method of claim 6 , wherein said one or more peptides or proteins comprises a detectable label.
8 . The method of claim 7 , wherein the detectable label is selected from the group consisting of a fluorescent label, nitrogen-15, carbon-13 and deuterium.
9 . The method of claim 1 , wherein said prostate cancer biomarker is FASN and comprises SEQ ID NO. 1.
10 . The method of claim 1 , wherein said affiliated molecular partner is USP2a and comprises SEQ ID NO. 2.
11 . The method of claim 1 , wherein the subject is a patient.
12 . The method of claim 1 , wherein the sample is serum.
13 . The method of claim 12 , wherein the sample obtained from the subject is treated to deplete at least one serum protein contained therein before digestion.
14 . The method of claim 1 , wherein the sample is obtained from said subject prior to administration to said subject of any treatment for cancer.
15 . The method of claim 1 , wherein the standard curve is generated having a lower data point at about 0.5 to 1.5 ng/mL protein concentration, an upper data point at 25 to 35 ng/mL protein concentration.
16 . The method of claim 15 , wherein the standard curve is generated using at least 3 data points within the range of about 1 to about 30 ng/mL protein concentration.
17 . The method of claim 15 , wherein the standard curve is generated having a lower data point at about 0.5 to 1.5 ng/mL protein concentration, an upper data point at 25 to 35 ng/mL peptide concentration, and at least 3 data points in the range of about 1 ng/mL to about 30 ng/mL peptide concentration.
18 . The method of claim 1 , wherein the standard curve has a maximum bias of no more than 20%.
19 . The method of claim 1 , where digestion is performed using trypsin.
20 . The method of claim 1 , where digestion is performed using endoproteinase Glu-C.
21 . The method of claim 1 , where digestion is performed using chymotrypsin.
22 . The method of claim 1 , wherein the mass spectrometry is MRM mass spectrometry with QQQ.
23 . A kit used to quantify the level of one or more prostate cancer biomarkers or affiliated molecular partners in a sample comprising two or more calibration standards selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 and combinations thereof.
24 . A synthetic isolated peptide 6 to 17 amino acids in length having at least 5 contiguous amino acids of a peptide selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43.
25 . The peptide of claim 23 where the peptide is from 6 to 17 amino acids in length.
26 . The peptide of claim 24 further comprising at least one detectable label.
27 . The peptide of claim 25 wherein the at least one detectable label is selected from the group consisting of a fluorescent label, nitrogen-15, carbon-13 and deuterium.
28 . The method of claim 1 , wherein the prostate cancer biomarker is NPY.
29 . The method of claim 2 , wherein the affiliated molecular partner is NPY.
30 . The method of claim 1 , wherein said prostate cancer biomarker is NPY and comprises SEQ ID NO. 3.
31 . The method of claim 1 , wherein said affiliated molecular partner is NPY and comprises SEQ ID NO. 3.
32 . The method of claim 1 , wherein said affiliated molecular partner is AMACR.
33 . The Method of claim 2 , wherein said affiliated molecular partner is AMACR.
34 . The method of claim 2 , wherein said affiliated molecular partner is AMACR and comprises SEQ ID NO. 4.
35 . The method of claim 28 , wherein said affiliated molecular partner is AMACR and comprises SEQ ID NO. 4.