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
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