Detection of paracetamol binding to albumin in blood serum using 2D-IR spectroscopy

Rutherford, Samantha H.; Greetham, Gregory M.; Towrie, Michael; Parker, Anthony W.; Kharratian, Soheila; Krauss, Thomas F.; Nordon, Alison; Baker, Matthew J.; Hunt, Neil T.

doi:10.1039/d2an00978a

Cited by 8 publications

(13 citation statements)

References 37 publications

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“…Such an observation has been associated with proteins becoming less dynamic upon ligand binding, leading to changes in anharmonicity. , By contrast, the impact of paracetamol binding to HSA yields a spectral change over an elongated frequency range. This is consistent with a recent study of paracetamol binding in equine serum and assigned also to anharmonicity changes . However, such spectral changes seen here may also be due to alterations of the diagonal line width …”

Section: Resultssupporting

confidence: 93%

“…Overall, this suggests that, in addition to identifying the presence of HSA-bound cefazolin in serum, the 2D-IR-derived data in Figure a can be used to quantify the binding behavior of cefazolin to serum proteins. This extends the findings of a previous study in which we used 2D-IR to quantify the level of paracetamol bound to equine serum albumin . Our new data show, in agreement with previous work, that cefazolin–serum interactions can be separated into two regimes.…”

Section: Resultssupporting

confidence: 91%

“…Recently, it has been shown that, unlike IR absorption, two-dimensional infrared spectroscopy (2D-IR) can measure protein amide I signals directly in biofluids by suppressing the water background, conferring the ability to measure protein concentrations and low-molecular-weight fractions of serum optically without sample preprocessing. − The nonlinear optical nature of the 2D-IR measurement confers slightly higher frequency resolution of the amide I band via narrower line widths than IR absorption spectroscopy, and it has been demonstrated that, in contrast to IR absorption methods, amide I band 2D-IR spectroscopy of serum albumin can be used to quantify levels of paracetamol binding at physiological concentrations in equine serum . Although this is an encouraging result, the promiscuous nature of albumin binding means that key questions remain related to whether the binding signature detected for paracetamol is specific to the identity of the drug molecule or a generic response of the protein to ligand binding and whether the effect is extendable to human serum.…”

Section: Introductionmentioning

confidence: 99%

“…The spectrum at T w = 250 fs contains protein signals free from the overlapping water band, whereas the spectrum acquired with T w = 5 ps contains a thermal water signal that is used in spectral preprocessing (see below). 15,23 For measurement of 2D-IR spectra, 10 μL of serum (see below for specific sample details) was placed between two CaF 2 windows without a spacer, and the tightness of the sample holder was adjusted to obtain a consistent absorbance of ∼0.1 for the δ H−O−H +υ libr combination mode of water located at 2130 cm −1 , corresponding to a sample thickness of ∼2.75 μm. 13 Drug-only control samples (see below) were measured using a sample path length of 25 μm, defined by a PTFE spacer.…”

Section: ■ Introductionmentioning

confidence: 99%

“…After 2D-IR data collection, spectral preprocessing was carried out for all serum samples using a previously described workflow designed to enable reliable cross-comparison of spectra obtained from different samples. 15 Briefly, the small thermal response of the water component of the samples measured using a T w of 5 ps was used to perform a bandwidth-guided baseline correction and normalization to account for instrumental and sample path-length variations between measurements. 23 Savitsky−Golay smoothing and principal component analysis noise reduction were also performed.…”

Section: ■ Introductionmentioning

confidence: 99%

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Optical Screening and Classification of Drug Binding to Proteins in Human Blood Serum

Rutherford,

Hutchison,

Greetham

et al. 2023

Anal. Chem.

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Protein−drug interactions in the human bloodstream are important factors in applications ranging from drug design, where protein binding influences efficacy and dose delivery, to biomedical diagnostics, where rapid, quantitative measurements could guide optimized treatment regimes. Current measurement approaches use multistep assays, which probe the protein-bound drug fraction indirectly and do not provide fundamental structural or dynamic information about the in vivo protein− drug interaction. We demonstrate that ultrafast 2D-IR spectroscopy can overcome these issues by providing a direct, label-free optical measurement of protein−drug binding in blood serum samples. Four commonly prescribed drugs, known to bind to human serum albumin (HSA), were added to pooled human serum at physiologically relevant concentrations. In each case, spectral changes to the amide I band of the serum sample were observed, consistent with binding to HSA, but were distinct for each of the four drugs. A machine-learning-based classification of the serum samples achieved a total cross-validation prediction accuracy of 92% when differentiating serum-only samples from those with a drug present. Identification on a per-drug basis achieved correct drug identification in 75% of cases. These unique spectroscopic signatures of the drug−protein interaction thus enable the detection and differentiation of drug containing samples and give structural insight into the binding process as well as quantitative information on protein−drug binding. Using currently available instrumentation, the 2D-IR data acquisition required just 1 min and 10 μL of serum per sample, and so these results pave the way to fast, specific, and quantitative measurements of protein−drug binding in vivo with potentially invaluable applications for the development of novel therapies and personalized medicine.

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