Quantitative Determination of Marker Proteins in Complex Mixtures

Background:
The Laboratory of Neurotoxicology of the National Institute of Mental Health and the Laboratory of Cell Biology of the National Cancer Institute are seeking statements of capability or interest from parties interested in forming a collaborative research alliance to further develop, evaluate, or commercialize techniques for protein quantification using mass spectrometry. This technology can be used in basic research, disease diagnosis or treatment to determine absolute and stoichiometric amounts of specific marker proteins in heterogeneous mixtures such as blood or urine.

High throughput methods have already been devised to purify cellular complexes from well-established cell models such as yeast using protein tags. Current techniques for protein quantification that utilize mass spectrometry depend on the quantification of peptides generated during proteolytic digestion. Chemical labeling of peptides after digestion or metabolic labeling of growing cells in the presence of labeled substrate are useful for relative analyses of different sample groups. Chemical synthesis of isotopically-labeled peptides has inherent limitations that may restrict proteotypic peptide selection, although complications have been circumvented by using a synthetic gene to code for a protein expressing a concatenated series of proteotypic peptides within a synthetic standard fusion protein. The approach was refined by incorporating flanking sequences into the fusion protein to account for sequence difference effects on proteolytic efficiency.

This technology utilizes a combination of recombinant DNA technology, fluorescence, proteolytic and mass spectrometric methods. The technique uses the identities of components known to be present in the mixture to construct and purify fusion proteins containing each of the proteins in the mixture fused to a protein exhibiting native fluorescence.

Value Proposition:

• Determination of protein stoichiometries, which is essential for determination of the three-dimensional structures of these complex mixtures.
• Enables automated quantification of proteins in a variety of laboratory, pharmaceutical and clinical settings where knowledge of absolute protein amounts is required.
• Provides, renewable protein standards for clinical analyses that can be regenerated.

Potential Application Areas:

• establish stoichiometries of proteins in cellular complexes involved in cell signaling;
• determine changes in stoichiometries as a function of cell cycle;
• determine absolute and stoichiometric amounts of specific marker proteins in heterogeneous mixtures such as blood or urine independent of antibody based assays or to calibrate antibody assays (for example, albumin/globulin ratio; pro-insulin/insulin/leptin ratio; clotting factor disorders);
• use as calibration standards for routine protein mass spectroscopy, including isotope dilution mass spectrometry;
• use as calibration standards for non-mass-spectrometric techniques where the relative molar response may vary, such as immunoassays, for example allowing use of Western blots for quantitative and stoichiometric measurements;
• determine the extent of post-translational modification of a signature peptide; the response for a post-translationally modified peptide will decrease relative to its known response factor.

R&D Status:
Pre-clinical validation. Demonstrated:

• Peak intensities obtained from tryptic peptide fragments have a high degree of reproducibility
• Method works using purified complexes of known stoichiometry,
• Absolute protein concentrations can be determined using purified fluorescent fusion proteins.

Challenges/Further R&D Needed:

• Design, construction, and testing of specific fusion proteins
• There remain significant options for fluorescent protein choices
• in vitro translation and isotope labeling methods need optimization

Related Publication(s):
Nanavati D, Gucek M, Milne JL, Subramaniam S, Markey SP. Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope labeled concatenated peptide standards. Mol Cell Proteomics. 2007 Nov 19.

Contact Information:
Sanford P. Markey, Ph.D.,
Senior Investigator
Laboratory of Neurotoxicology
National Institute of Mental Health
E-mail: markeys@mail.nih.gov

Reference:  #586 KH

Posted 12/06/2007