Detection of LARP1
View Patent ↗The invention relates to the detection of EGYR peptide in a biological sample as a measure of the presence and/or amount of LARP1 protein in the sample. Suitably, the invention relates to methods for quantitative measurement of LARP1 and LARP1-derived EGYR peptide by chromatography-tandem mass spectrometry. The invention also relates to peptide standards and their use in quantitative mass spectrometric analyses. The ability to detect the amount of LARP1 in a biological sample has application in detecting and monitoring cancer.
1. A method for detecting LARP-1 protein in a biological sample, comprising producing a protein digest from the biological sample; and detecting for the presence of EGYR fragment peptide in the protein digest, thereby determining the presence of LARP-1 in the biological sample.
2. The method of claim 1 , wherein the protein digest comprises a serine-protease digest.
3. The method of claim 2 , wherein the serine-protease digest comprises a trypsin digest.
4. The method of claim 1 , wherein the proteins in the sample are reduced and alkylated.
5. The method of claim 1 , wherein the detecting comprises mass spectrometry (MS).
6. The method of claim 5 , wherein the EGYR peptide is fragmented in tandem mass spectrometry (MS: MS) mode and a fragment of EGYR with m/z of 136.1 in positive ion mode ionisation is detected.
7. The method of claim 1 , wherein the detecting comprises liquid chromatography (LC) and MS.
8. The method of claim 1 , wherein the biological sample is selected from the group consisting of: a blood sample, a plasma sample, a serum sample, a urine sample, an ascites sample and a saliva sample.
9. The method of claim 1 , wherein the biological sample is from a patient with or suspected of having cancer or pre-cancer.
10. The method of claim 9 , wherein the cancer is selected from the group consisting of: ovarian cancer, breast cancer, pancreatic cancer, colon cancer and hepatocellular cancer.
11. The method of any of claim 9 , wherein the patient possesses germline BRCA1 or BRCA-2 mutations or other cancer-predisposing mutations/syndromes.
12. The method of claim 1 , further comprising further quantifying the EGYR fragment peptide.
13. The method of claim 12 , wherein quantifying the EGYR fragment peptide comprises comparing the amount of detected peptide to an EGYR peptide standard of known amount, wherein the EGYR peptide standard is isotopically labelled.
14. A method of predicting the presence of serous tubal intraepithelial carcinoma (STIC) in a subject comprising detecting the level of LARP1 protein in a serum or plasma sample from the subject; and comparing the level of LARP1 protein in said sample with a reference value of LARP1 protein or with the level of LARP1 in a control sample, wherein an elevated level of LARP1 protein in said sample from said subject compared to the reference value or the level of LARP1 protein in the control sample predicts that the subject has STIC; wherein the level of LARP1 protein is quantified by detecting the level of LARP1-derived EGYR peptide.
15. A method for diagnosing or predicting risk of developing cancer, the method comprising:
subjecting a biological sample obtained from a patient to one or more processing steps to cleave the proteins in the sample into smaller peptides, wherein if LARP1 protein is in the sample the peptides produced include EGYR peptide;
subjecting the processed sample to ionization under conditions suitable to produce one or more multiply charged ions detectable by mass spectrometry;
determining by mass spectrometry the amount of one or more ions from the LARP1-derived EGYR peptide; and
using the determined ion amounts to determine the amount of LARP1 protein in the biological sample or EGYR peptide in the processed sample, wherein an elevated LARP1 protein level in the biological sample relative to normal indicates the presence of or risk of developing cancer.
16. The method according to claim 15 , wherein the patient comprises cancer-predisposing mutations in its genomic DNA.
17. The method according to claim 16 , wherein the patient possesses germline BRCA1 or BRCA-2 mutations.
18. The method according to claim 16 , wherein the patient comprises germline driving mutations in a gene selected from: BRCA1, BRCA2, KRAS, P53, ALK and HER2.
19. The method according to claim 15 , wherein the patient has a syndrome selected from BRCA, Lynch and Li Fraumen.
20. A peptide standard for quantifying LARP-1 in a biological sample, comprising an amino acid sequence EGYR, wherein said peptide standard is utilized in the method of claim 1 .
21. The peptide standard of claim 20 , wherein the arginine amino acid in the EGYR peptide is labelled with an isotope containing four 15N and six 13C, leading to a mass shift of +10 Da.
22. The method according to claim 14 , wherein the subject comprises cancer-predisposing mutations in its genomic DNA.
23. The method according to claim 22 , wherein the patient possesses germline BRCA1 or BRCA-2 mutations.
24. The method according to claim 22 , wherein the patient comprises germline driving mutations in a gene selected from: BRCA1, BRCA2, KRAS, P53, ALK and HER2.