Toward Absolute\nQuantification of Soluble Proteins\nvia Proton Nuclear Magnetic Resonance Spectroscopy: Total Protein\nConcentration in Blood Plasma

For absolute protein quantification using nuclear magnetic\nresonance\n(NMR) spectroscopy, we considered proteins as homopolymers and effective\namino acid (AA) residues (AAR<sub>Eff</sub>) as monomer units. For\ndiverse classes of proteins, we determined the AAR<sub>Eff</sub> molecular\nweight as 111.5 ± 3.2 Da and the number of hydrogens per AA as\n7.8 ± 0.2. Their ratio of 14.3 ± 0.3 (g/LP)/(mol/LH) remains\nconstant across various protein classes and is equivalent to Kjeldahl’s\nnitrogen-to-protein conversion constant of 5.78 ± 0.29 gN/gP.\nBy analogy to the Kjeldahl method, we suggest that the total integral\nof a <sup>1</sup>H NMR solution protein spectrum could be used for\ntotal protein quantification. We synthesized low-resolution protein\nspectra from the weighted sums of individual AA spectra and compared\nthem with experimental spectra. In the methyl region, the ratio of\nthe protein mass to the total number of protons in the synthetic spectra\n(corrected for the chemical shift mismatch) was ∼1 (mg/mL)/mM,\nwhich agrees with an earlier reported experimental ratio for urine\n(1.05 ± 0.06 (mg/mL)/mM). For human blood plasma, in the methyl\nregion, we found empirical ratios of 1.115 ± 0.006 (mg/mL)/mM\n(using 96 patient samples) and 1.121 ± 0.011 (mg/mL)/mM for the\nNIST plasma standard. This numerical agreement points to universal\nconversion constants, i.e., protein mixtures with unknown compositions\ncould be quantified without the need for calibration standards by\nmeasuring the millimolar proton concentration within the methyl region\nof the NMR spectrum using the same conversion constant.