Chemometric approaches to enhance the potential of new IR spectroscopic technologies

GOICOECHEA, HÉCTOR CASIMIRO; ALCARAZ, MIRTA RAQUEL; AKHGAR, CHRISTOPHER K.; SCHWAIGHOFER, ANDREAS; EBNER, JULIAN; LENDL, BERNHARD

Roma

Conferencia; 18th Chemometrics in Analytical Chemistry Conference; 2022

It is well demonstrated that alternating least square (ALS)-based approaches significantly aid in comprehensively understanding the behaviour of a system under study due to the possibility of obtaining meaningful results with physical and chemical interpretation. This tool has been vastly exploited, particularly in the spectroscopy field, aiming to enhance the efficiency of experimental studies. Notably, applications based on novel instrumental technologies are profiting from its advantages encouraging a mutual broadening of their capacities. Here, the benefits of the usage of ALS-based methodologies in quantum cascade laser-infrared (QCL-IR) spectroscopic-based applications for protein analysis are described. Mid-IR spectroscopy is a well-established analytical technique routinely employed to study the structure of polypeptides and proteins in a label-free manner. However, the low feasible path lengths needed in conventional Fourier transform (FT)-IR spectrometers for IR transmission measurements of proteins in aqueous solutions are a considerable impairment for the robustness of analysis and impede flow-through measurements and high-throughput applications. In this regard, a new technology based on QCLs emerges as a light source highly interesting for measurements of aqueous samples [1]. Notwithstanding, their applicability in protein analysis in dynamic processes is a hot research topic. First, a QCL-based setup for mid-IR transmission measurements in the protein amide I region was used for monitoring dynamic changes in the secondary structure of proteins. α-Chymotrypsin acts as a model protein, which gradually forms intermolecular β-sheet aggregates after adopting a non-native α-helical structure induced by exposure to 50% TFE. The effects of varying pH values and protein concentration on the rate of β-aggregation were studied. Extended multivariate curve resolution (MCR)-ALS was employed to obtain pure spectral and concentration profiles of the temporal transition between α-helices and intermolecular β-sheets. The results demonstrated the potential and versatility of the QCL-based IR transmission setup to monitor dynamic changes of protein secondary structure in aqueous solutions coupled with chemometric analysis [1].Second, a different QCL setup for mid-IR transmission spectroscopy in the amide I and II region was used for monitoring pH-induced changes in the secondary structure of -lactoglobulin. Chemometric analysis of the dynamic IR spectra was performed by MCR-ALS. Then, a postprocessing procedure based on wavelet analysis was implemented to extract information about protein spectra and spurious signals that may interfere with the result interpretation [2]. Last, a QCL-based flow-through mid-IR spectrometer was placed in-line with a preparative size exclusion chromatography (SEC) system to demonstrate real-time analysis of protein elution with overlapping chromatographic peaks. Chemometric analysis by self-modelling mixture analysis (SMMA) and MCR enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in agreement with off-line liquid chromatography (HPLC) reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors in combination with chemometrics can be used effectively for in-line, real-time analysis of protein elution. All these works shed light on the fact that EC-QCL-based IR transmission setup combined with chemometric analysis has high potential and versatility for dynamic and flow-through applications.