Isotopically-labeled proteome standards
The invention provides methods for quantifying biomolecules, such as polypeptides in mass spectrometric analysis. The methods include use of a biomolecule standard having at least one atomic isotope different than that of the naturally occurring isotopes in the biomolecule of interest. Methods of the present invention also include methods for quantifying biomolecules where the copy biomolecule standard is made by expressing the biomolecule using a recombinant cell. Further included are the biomolecule standards themselves, method for making such standards, kits, systems, reagents, and engineered cells relating to the use of biomolecule standards in mass spectrometric analysis.
1. A method of making an isotopically-labeled biomolecule standard comprising
expressing a copy of a protein of interest in recombinant cells cultured in media that comprises at least one isotopically-labeled amino acid or amino acid residue, wherein the expressed copy of the protein incorporates the at least one isotopically-labeled amino acid or amino acid residue;
expressing a cognate protein of interest expressed in the recombinant cells cultured in media comprising at least one naturally-occurring cognate amino acid or amino acid residue in the absence of the at least one isotopically-labeled amino acid or amino acid residue, wherein the cognate protein of interest comprises the at least one naturally-occurring cognate amino acid or amino acid residue;
purifying the copy of the protein of interest, wherein the purified copy of the protein is not enzymatically or chemically digested, wherein the undigested, purified copy of the protein is used as an isotopically-labeled biomolecule standard; and
quantitating the isotopically-labeled biomolecule standard by comparing the mass spectrometry peaks of pairs resulting from the isotopically-labeled biomolecule standard and the cognate protein of interest, wherein the mass of the isotopically-labeled biomolecule standard differs from the mass of the cognate protein of interest by an amount corresponding to the difference in mass between the isotopic label and the naturally-occurring cognate.
2. The method of claim 1 , wherein the at least one isotoptically-labeled amino acid or amino acid residue comprises a non-radioactive isotope selected from the group consisting of 15 N, 13 C, 18 O, 2 H, 34 S, and combinations thereof.
3. The method of claim 1 , wherein the quantity being determined is a rely quantity.
4. The method of claim 1 , wherein die quantity being determined is an absolute quantity.
5. The method of claim 1 , wherein the cultured cells are prokaryotic cells.
6. The method of claim 5 , wherein the prokaryotic cells are selected from the group consisting of E. coli cells, Pseudomonas cells, Rhodobacter cells, Bacillus cells, Streptomyces cells, Erwinia cells, Klebsiella cells, Serratia cells and Salmonella cells.
7. The method of claim 1 , wherein the cells are eukaryotic cells.
8. The method of claim 1 , wherein the cells are selected from the group consisting of yeast cells, mammalian cells, marsupial cells, avian cells, insect cells, fish cells, amphibian cells, reptile cells, and nematode cells.
9. The method of claim 1 , wherein the copy is made using at least one amino acid with an isotope pool having a mass distribution different than that of naturally-occurring isotopes in the cognate protein of interest.
10. The method of claim 1 , wherein the amino acid is selected the group consisting of Arg, Lys, Asp, GIu, Met, Trp, Ser, Thr, Tyr, and Asn.