The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems.
Glycine (Gly) is used as a model system to evaluate the ability of ultrafast 2D-IR spectroscopy to detect and quantify the low-molecular weight proteinaceous components of blood serum. Combining data acquisition schemes to suppress absorption bands of H2O that overlap with the protein amide I band with analysis of peak patterns appearing in the off-diagonal region of the 2D-IR spectrum allows separation of the Gly spectral signature from that of the dominant protein fraction of serum in a transmissionmode 2D-IR measurement without any sample manipulation, e.g. filtration or drying. 2D-IR spectra of blood serum samples supplemented with varying concentrations of Gly were obtained and a range of data analysis methods compared, leading to a detection limit of ~ 3 mg/mL for Gly. The reported methodology provides a platform for a critical assessment of the sensitivity of 2D-IR for measuring the concentrations of amino acids, peptides and low molecular weight proteins present in serum samples. We conclude that, in the case of several clinically relevant diagnostic molecules and their combinations, the potential exists for 2D-IR to complement IR absorption methods as the benefits of the second frequency dimension offered by 2D-IR spectroscopy outweigh the added technical complexity of the measurement.
The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H2O- rather than D2O-based solvents by evading interfering water signals has enabled in-vivo studies of proteins under physiological conditions and in biofluids . Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will however require comparisons between multiple datasets, necessitating control of data collection protocols to minimise measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalisation strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously-published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress towards the robust data handling strategies that will be necessary for future applications of 2D-IR for pharmaceutical or biomedical applications.
Biofluid spectroscopy is an emerging technology in the field of clinical investigation, providing a simple way to extract diagnostic and observational information from easy to acquire samples. Infrared spectroscopy is well suited to analyse a large range of biofluid samples, including blood and its derivatives, due to flexible sampling modes and high sensitivity to subtle biological changes. As the technology advances towards the clinic, factors influencing successful clinical translation are becoming apparent. Here, we provide a tutorial for effective biofluid spectroscopy study design, discussing sample and instrument parameters, as well as clinical considerations. The aim is to present the current understanding of clinical translation in the field of biofluid spectroscopy, and to facilitate other clinical applications to advance to the clinic.
Two-Dimensional Infrared (2D-IR) spectroscopy is used to detect binding of paracetamol with proteins in blood serum. Quantitative peak patterns are observed indicating structural changes of the albumins' secondary structure when paracetamol bound.
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