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Measuring proteins in H2O with 2D-IR spectroscopy


The amide I infrared band of proteins is highly sensitive to secondary structure, but studies under physiological conditions are prevented by strong, overlapping water absorptions, motivating the widespread use of deuterated solutions. H/D exchange raises fundamental questions regarding the impact of increased mass on protein dynamics, while deuteration is impractical for biomedical or commercial applications of protein IR spectroscopy. We show that 2D-IR spectroscopy can avoid this problem because the 2D-IR amide I signature of proteins dominates that of water even at sub-millimolar protein concentrations. Using equine blood serum as a test system, we investigate the significant implications of being able to measure the spectroscopy and dynamics of proteins in water, demonstrating relevance in areas ranging from fundamental science to the clinic. Measurements of vibrational relaxation dynamics of serum proteins reveals that deuteration slows down the rate of amide I vibrational relaxation by > 10%, indicating a dynamic impact of isotopic exchange in some proteins. The unique link between protein secondary structure and 2D-IR amide I lineshape allows differentiation of signals due to albumin and globulin protein fractions in serum leading to measurements of the biomedically-important albumin to globulin ratio (AGR) with an accuracy of ±4% across a clinically-relevant range. Furthermore, we demonstrate that 2D-IR spectroscopy enables differentiation of the structurally-similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing.

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Publication details

The article was received on 01 Apr 2019, accepted on 13 May 2019 and first published on 14 May 2019

Article type: Edge Article
DOI: 10.1039/C9SC01590F
Chem. Sci., 2019, Accepted Manuscript
  • Open access: Creative Commons BY-NC license
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    Measuring proteins in H2O with 2D-IR spectroscopy

    S. Hume, G. Hithell, G. M. Greetham, P. Donaldson, M. Towrie, A. W. Parker, M. J. Baker and N. Hunt, Chem. Sci., 2019, Accepted Manuscript , DOI: 10.1039/C9SC01590F

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