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Metallomics:  A Multidisciplinary Approach for the Qualitative and Quantitative Determination of Proteins and Metalloproteins Used as Health and Disease Markers

Summary:


As healthcare shifts from management of disease to management of health, there will be a necessary shift toward development of quantitative health information. This will require a parallel metrological effort directed toward the development of quantitative and sensitive analytical methods to detect protein biomarkers, metabolites, and trace elements in clinical samples. By performing cutting edge, multidisciplinary research on metals associated with proteins, NIST will continue to stay at the forefront with regard to benchmarking chemical measurements important to the healthcare sector.

Description:


The emerging science of “metallomics,” which involves the detection, mapping, and/or quantification of trace elements in biological systems at organ, cell, and subcell levels, requires the development of versatile measurement tools allowing for trace detection, identification, and quantification of metals associated with proteins. The goal of this project is to improve the state of the practice for protein metrology through development of analytical methods for separation, detection and quantification of proteins in biological tissues and fluids.

Additional Technical Details:


The complexation and binding of metals to proteins creates challenges in developing analytical methods because these associations rarely involve covalent bonding. Metals can be easily lost when protein separations are performed using either gel electrophoresis or various forms of liquid chromatography, and even during initial sample cleanup procedures. Selenium is an exception, and selenoproteins are good candidates to develop and test methods for protein isolation, separation, and quantification using inductively coupled plasma mass spectrometry (ICP-MS) as a selective and sensitive detection platform. Newer technologies are emerging, such as metal-coded affinity tagging (MeCAT) of proteins, whereby the metal becomes a surrogate for measuring the protein. This tagging technique is under investigation for application to the absolute quantification of biomarker proteins that would otherwise be undetectable in ICP-MS. 

Research has begun on developing new quantification methods for metalloproteins using the associated metal. Experiments have been performed to synthesize isotopic metal analogues of copper metalloproteins through dialytic exchange and employing them as calibrants for isotope dilution mass spectrometry. Progress has been made using antibody pull-down protein purification methods to isolate and preconcentrate C-reactive protein (CRP), a calcium binding protein and inflammatory marker. Advances have been made in method development involving detection of selenoproteins (Figure 1), and laser ablation (LA) ICP-MS is being investigated for metal screening of separated proteins on polyacrylamide gels. We are investigating metal coded affinity tagging (MeCAT) technology, which enables elemental labeling of any protein, as a complement to methods that require careful preservation of the native metal cofactor associated with metalloproteins. This technology relies on attaching small, bifunctional chemical tags to proteins that remain covalently attached to protein functional groups, while strongly chelating a lanthanide metal, allowing the metal-tagged proteins to survive harsh analytical method steps. MeCAT technology has the potential to improve protein quantification for all classes of proteins, including metalloproteins. ICP-MS may ultimately provide for ultra-sensitive quantification of metal-containing proteins or peptides that can be accurately determined down to the low attomole range; detection limits that are at least 2 to 3 orders of magnitude lower than other complementary mass spectrometric techniques.

Major Accomplishments:

  • Development of LC/ICP-MS methods for the separation of selenium species, including selenoproteins in liver and blood    
  • Proof-of-principle isotope dilution experiments have been performed after synthesis of isotopic copper protein calibrants     
  • Methods and techniques have been implemented for coupling 2-D and 1-D gel electrophoresis to laser ablation ICP-MS for detection of metal-containing proteins   
  • Development of methods for isolation of C-reactive protein using antibody pull-down methods.