Methods and systems for the analysis of protein samples

1. A tangible computer readable medium storing instructions capable of being executed on a processor so as to perform a method for detecting the presence of a mutation, modification, or impurity in a sample of a protein of interest, the method comprising:

measuring, using a mass spectrometer, a mass spectrum of intensity as a function of mass to charge ratio (m/z) of peptides obtained from a digested mixture of:

a first reference sample consisting essentially of a single protein of interest having a known amino acid sequence, wherein at least one amino acid in the protein of interest is replaced with an isotopically labeled amino acid comprising at least one heavy isotope; and

a second sample of the single protein of interest comprising an unlabeled amino acid corresponding to the isotopically labeled variant in the reference protein;

wherein substantially all peptides of the reference sample in the digest comprise at least one isotopically labeled amino acid; and

wherein the first labeled reference sample of the protein of interest and the second unlabeled sample of the protein of interest are mixed in a 1:1 ratio prior to digestion;

wherein the m/z values corresponding to the mass spectrum of peptides cover more than 80% of the sequence of the protein of interest;

determining m/z values within the mass spectrum corresponding to each monoisotopic intensity peak;

identifying those m/z values that correspond to peptides comprising an isotopically labeled amino acid;

identifying the presence of each doublet within the mass spectrum, wherein each doublet indicates the presence of a peptide with an isotopically labeled amino acid and a corresponding peptide without an isotopically labeled amino acid; and

determining an intensity ratio for each identified doublet in the mass spectrum from the ratio of the peak intensity of the m/z value for the unlabeled peptide in the second unlabeled sample of the protein of interest to the m/z value for the corresponding peptide in the first labeled reference sample of the protein of interest; and

detecting the presence of a mutation, modification, or impurity in the second unlabeled sample of the protein of interest when the intensity ratio for any doublet is less than one.

2. A tangible computer readable medium storing instructions capable of being executed on a processor so as to perform a method for detecting the presence of a mutation, modification, or impurity in a sample of a protein of interest, the method comprising:

measuring, using a mass spectrometer, a first mass spectrum of intensity as a function of mass to charge ratio (m/z) of peptides obtained from a first digested mixture of:

a first reference sample consisting essentially of a single protein of interest having a known amino acid sequence, wherein at least one amino acid in the reference protein is replaced with an isotopically labeled amino acid comprising at least one heavy isotope; and

a second sample of the single unlabeled protein of interest comprising an unlabeled amino acid corresponding to the isotopically labeled variant in the reference protein;

wherein substantially all peptides of the reference protein in the digest comprise at least one isotopically labeled amino acid; and

wherein the first labeled reference sample of the protein of interest and the second unlabeled sample of the protein of interest are mixed in a 1:1 ratio prior to digestion;

wherein the m/z values corresponding to the first mass spectrum of peptides cover more than 80% of the sequence of the protein of interest;

determining m/z values within the first mass spectrum corresponding to each monoisotopic intensity peak;

identifying those m/z values within the first mass spectrum that correspond to peptides comprising an isotopically labeled amino acid;

identifying the presence of each doublet within the first mass spectrum, wherein each doublet indicates the presence of a peptide with an isotopically labeled amino acid and a corresponding peptide without an isotopically labeled amino acid; and

determining a first intensity ratio for each identified doublet in the first mass spectrum from a ratio of the peak intensity of the m/z values for the unlabeled peptide in the second unlabeled sample of the protein of interest to the peak intensity of the m/z value for the corresponding peptide in the first labeled reference sample of the protein of interest;

measuring, using a mass spectrometer, a second mass spectrum of intensity as a function of mass to charge ratio of peptides obtained from a second digested mixture of:

the first labeled reference sample of the protein of interest; and

a third sample of the single protein of interest comprising an unlabeled amino acid corresponding to the isotopically labeled variant in the reference protein;

wherein the first labeled reference sample of the protein of interest and the third unlabeled sample of the protein of interest are mixed in a 1:1 ratio prior to digestion;

wherein the m/z values corresponding to the second mass spectrum of peptides covers more than 80% of the sequence of the protein of interest;

determining m/z values within the second mass spectrum corresponding to each monoisotopic intensity peak;

identifying those m/z values that correspond to peptides comprising an isotopically labeled amino acid;

identifying the presence of each doublet within the second mass spectrum, wherein each doublet indicates the presence of a peptide with an isotopically labeled amino acid and a corresponding peptide without an isotopically labeled amino acid;

determining a second intensity ratio for each identified doublet in the second mass spectrum from a ratio of the peak intensity of the m/z values for the unlabeled peptide in the third unlabeled sample of the protein of interest to the peak intensity of the m/z value for the labeled peptide in the first labeled reference sample of the protein of interest;

determining a reference ratio from a ratio of the first intensity ratio to the second intensity ratio; and

detecting the presence of a mutation, modification, or impurity in the first sample of the protein of interest when the reference ratio for any doublet is less than one.

3. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 wherein the m/z values corresponding to the mass spectrum of peptides cover more than 90% of the sequence of the protein of interest.

4. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 wherein the m/z values corresponding to the mass spectrum of peptides cover more than 95% of the sequence of the protein of interest.

5. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 wherein the m/z values corresponding to the mass spectrum of peptides cover the entire sequence of the protein of interest.

6. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 , further comprising obtaining:

a list of retention times corresponding to the mass spectrum.

7. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining:

a list of retention times corresponding to the first mass spectrum.

8. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 , further comprising obtaining an error tolerance/deviation for the m/z and the retention time values.

9. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 , further comprising obtaining a minimum intensity that is allowed for the m/z values.

10. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 1 , further comprising obtaining a maximum intensity that is allowed for the m/z values.

11. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the first mass spectrum of peptides cover more than 90% of the sequence of the protein of interest.

12. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the first mass spectrum of peptides cover more than 95% of the sequence of the protein of interest.

13. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the first mass spectrum of peptides cover the entire sequence of the protein of interest.

14. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the second mass spectrum of peptides cover more than 90% of the sequence of the protein of interest.

15. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the second mass spectrum of peptides cover more than 95% of the sequence of the protein of interest.

16. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 wherein the m/z values corresponding to the second mass spectrum of peptides cover the entire sequence of the protein of interest.

17. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining a list of retention times corresponding to the second mass spectrum.

18. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining an error tolerance/deviation for the m/z and the retention time values for the first mass spectrum.

19. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining an error tolerance/deviation for the m/z and the retention time values for the second mass spectrum.

20. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining a minimum intensity that is allowed for the m/z values of the first mass spectrum.

21. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining a minimum intensity that is allowed for the m/z values of the second mass spectrum.

22. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining a maximum intensity that is allowed for the m/z values of the first mass spectrum.

23. The tangible computer readable medium storing instructions capable of being executed on a processor of claim 2 , further comprising obtaining a maximum intensity that is allowed for the m/z values of the second mass spectrum.