US 7,125,685 B2 · Stable isotope, site-specific mass tagging for protein identification

Abstract

Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

Claims30

Dependent claims

A method for identifying a protein, the method comprising the steps of:

(a) separating the protein from other proteins;
(b) digesting the protein, thereby forming first proteolytic peptides;
(c) acquiring a monoisotopic mass distribution spectrum of the first proteolytic peptides and acquiring the m/z values for the first proteolytic peptides;
(d) introducing at least one amino acid 100% labeled with at least one stable isotope into a cell that expresses a protein, and wherein the cell is induced to express the protein comprising the at least one amino acid 100% labeled in a sequence-specific manner;
(e) separating the protein bearing the at least one labeled amino acid from other proteins;
(f) digesting the protein bearing the at least one labeled amino acid, thereby forming second proteolytic peptides;
(g) acquiring the monoisotopic mass distribution spectrum of the second proteolytic peptides and acquiring the m/z values for the first proteolytic peptides;
(h) comparing the monoisotopic mass distribution spectrum of the second proteolytic peptides with the monoisotopic mass distribution spectrum of the first proteolytic peptides to determine the amino acid composition of the first proteolytic peptides and the second proteolytic peptides, whereby the protein is identified from the m/z values of the first proteolytic peptides and the m/z values of the second proteolytic peptides and the amino acid composition of the first proteolytic peptides and the second proteolytic peptides.

5 more elements

Dependencies are inferred from the claim text.

Stable isotope, site-specific mass tagging for protein identification

Life of this patent

Stable isotope, site-specific mass tagging for protein identification

Life of this patent

Abstract

Claims30