Analyte mass spectrometry quantitation using a universal reporter
Quantitation of analytes, including but not limited to peptides, polypeptides, and proteins, in mass spectrometry using a labeled peptide coupled to a reporter, and a universal reporter.
1. A mass spectrometry (MS) analyte quantitation method comprising
preparing from a sample containing an analyte, the analyte having a known defined sequence, a heavy isotopically labeled sequence or part thereof that fragments into a predictable ion series following MS dissociation to specifically identify and quantitate the analyte, the sequence cleavably linked to a reporter R sequence;
providing a heavy isotopically labeled universal reporter U having a sequence identical to the reporter R sequence but independent from the analyte sequence, where universal reporter U and reporter R have a different mass;
effecting cleavage, and
quantitating universal reporter U to quantitate reporter R, which is in equimolar concentration to the heavy isotopically labeled analyte, thereby determining the quantity of analyte in the sample.
2. A mass spectrometry method comprising quantitating a proteotypic peptide in a sample using an isotopically labeled universal reporter peptide U having a peptide sequence that is identical to a reporter peptide R sequence but independent from the sequence of the proteotypic peptide, where universal reporter U and reporter R have a different mass; cleaving a heavy isotope labeled proteotypic peptide cleavably linked to the reporter peptide R; and using the universal reporter peptide U to quantitate reporter peptide R, which is in equimolar concentration to the heavy isotope labeled proteotypic peptide, to determine the quantity of proteotypic peptide in the absence of amino acid analysis.
3. The method of any of claims 1 - 2 performed in a single reaction with at least two concatenated proteotypic peptides.
4. The method of any of claims 1 - 2 performed in a single reaction with at least three concatenated proteotypic peptides as in FIG. 4 .
5. The method of any of claims 1 - 2 performed in a single reaction with at least two proteotypic peptides, each proteotypic peptide cleavably linked to a different reporter peptide R and with a single universal reporter peptide U.
6. The method of any of claims 1 - 2 performed in a single reaction with at least three proteotypic peptides, each proteotypic peptide cleavably linked to a different reporter peptide R and with a single universal reporter peptide U as in FIG. 5 .
7. The method of claim 1 wherein universal reporter U is a polymer.
8. The method of any of claims 1 - 2 wherein the universal reporter U is added to the sample before the cleavage step.
9. The method of any of claims 1 - 2 wherein the universal reporter U is added to the sample after the cleavage step.
10. The method of claim 1 further comprising adding at least one component that enhances water solubility of the analyte.
11. The method of any of claims 1 - 2 further comprising adding at least one component that enhances detection of the analyte.
12. The method of any of claims 1 - 2 further comprising adding at least one component that enhances stability of the analyte.
13. The method of any of claims 1 - 2 further comprising labeling the universal reporter U and the reporter R with a fluorophore and/or chromophore, and measuring the absorbance of the fluorophore and/or chromophore.
14. The method of any of claims 1 - 2 wherein the universal reporter U contains at least one tryptophan, and universal reporter U is quantified by measuring absorbance using the specific extinction factor of tryptophan.
15. The method of claim 10 using the sequence PVVVPR (SEQ ID NO. 1) or the sequence LVALVR (SEQ ID NO. 52) as reporter peptide R for enhancing solubility.
16. The method of any of claims 1 - 2 performed in a multiplex assay.
17. The method of any of claims 1 - 2 wherein reporter R contains a heavy isotope label.
18. The method of any of claims 1 - 2 wherein reporter R lacks a heavy isotope label.
19. The method of claim 2 further comprising adding at least one component that enhances water solubility of the analyte.
20. The method of claim 19 using the sequence PVVVPR (SEQ ID NO. 1) or the sequence LVALVR (SEQ ID NO. 52) as reporter peptide R for enhancing solubility.