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 kit comprising at least one amino acid containing two or more atomic isotopes that are different in molecular mass from naturally-occurring isotopes, wherein the at least one amino acid is not incorporated into a peptide or protein; and one or more of:
a protein synthesis system;
a template instruction for synthesizing a biomolecule standard;
a resin for purifying said biomolecule standard;
a protein quantitation reagent;
an enzyme or chemical reagent capable of cleaving said biomolecule standard;
an enzyme or chemical preparation capable of cleaving said biomolecule standard;
a vector; and
a polymerase.
2. The kit of claim 1 , wherein said synthesis system comprises an extract selected from the group consisting of a bacterial extract, a eukaryotic extract, a plant extract, a mammalian extract, and an insect extract.
3. The kit of claim 1 , further comprising at least one buffer for in vitro protein synthesis.
4. The kit of claim 1 , wherein at least one isotope of said two or more atomic isotopes is an atom selected from the group consisting of 15 N, 13 C, 18 O, 2 H and 34 S.
5. The kit of claim 1 , wherein said enzyme is selected from the group consisting of trypsin, Endo-Lys-C, Endo-Glu-C, AspN protease, a yeast peptidase, V-8 protease, pepsin, subtilisin, proteinase lc, tobacco etch virus protease, and an enzyme with cleavage characteristics similar to trypsin.