Measuring Protein Concentration by Diffusion-Filtered Quantitative Nuclear Magnetic Resonance Spectroscopy

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biophysics

Background:

• Accurate macromolecule concentration is vital for biological therapeutics.
• Existing protein concentration methods lack a priori molecular property detection.
• Current methods rely on indirect measurements or experimentally determined properties.

Purpose of the Study:

• To introduce a diffusion-filtered quantitative NMR (DF-qNMR) experiment for macromolecule quantitation.
• To present an overall method for absolute protein concentration measurement.
• To validate the VILMHA method for protein quantitation in complex matrices.

Main Methods:

• Developed a diffusion-filtered qNMR (DF-qNMR) experiment.
• Combined protein denaturation with DF-qNMR for clean spectra.
• Quantitated proteins by comparing valine/isoleucine/leucine methyl group peak areas to a certified standard.
• Referred to the method as VILMHA (valine isoleucine leucine methyl hydrogen analysis).

Main Results:

• VILMHA measured protein concentrations within 1.8% of labeled values for reference materials.
• Relative Standard Deviations (RSDs) were consistently below 1.25%, often under 1%.
• The method demonstrated high accuracy, precision, and ease of use across various protein sizes.

Conclusions:

• VILMHA provides a superior method for absolute protein concentration determination compared to existing techniques.
• DF-qNMR and VILMHA can convert relative protein concentration methods to absolute ones.
• DF-qNMR has potential for quantitating other macromolecules like polymers and surfactants.