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Measuring Protein Concentration by Diffusion-Filtered Quantitative Nuclear Magnetic Resonance Spectroscopy
journal contribution
posted on 2019-01-04, 00:00 authored by Scott A. Bradley, Wesley C. Jackson, Patrick P. MahoneyThe
concentration of macromolecules in solution is a crucial property
in many areas of research, including the development and commercialization
of biological therapeutics. For proteins in particular, none of the
reported methods for measuring concentration detect a molecular property
that is known a priori; rather, they rely on ligand binding, degradation
and derivitization, or an intrinsic property that must be determined
experimentally. The purpose of this report is to describe (1) a diffusion-filtered
qNMR experiment (DF-qNMR) for quantitating macromolecules in complex
matrices and (2) an overall method for measuring absolute protein
concentration based on this DF-qNMR experiment. This method combines
protein denaturation with the diffusion filter to produce clean spectra
of the protein with well-resolved resonances, regardless of the matrix
complexity. The concentration is then obtained by comparing the peak
area of the valine/isoleucine/leucine methyl groups to an external,
certified, small-molecule quantitation standard. The method, which
is referred to as VILMHA (valine isoleucine leucine methyl hydrogen
analysis), was tested on three proteins of various sizes. In all cases,
the measured concentration was within 1.8% of the labeled value for
the undiluted standard reference material evaluated. In addition,
the RSD’s were less than 1.25% in all cases and less than 1%
in most cases. The accuracy, precision, and ease of use make this
method superior to existing absolute protein concentration methods.
Furthermore, VILMHA is ideally suited to serve as the basis for converting
the relative protein concentration methods into absolute methods or
establishing molecular-specific parameters. Finally, DF-qNMR has the
potential to quantitate other types of macromolecules (e.g., such
as polymers, surfactants, etc.) in the presence of small-molecule
contaminants.
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Keywords
Resonance Spectroscopyprotein concentration methodsDF-qNMR experimentligand bindingwell-resolved resonancesdiffusion-filtered qNMR experimentprotein denaturationmolecular-specific parameterssmall-molecule quantitationmatrix complexityDiffusion-Filtered Quantitative NuclearVILMHAsmall-molecule contaminantsquantitating macromoleculesreference materialdiffusion filterProtein Concentrationpeak areaRSDprotein concentration
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