Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and C H stretching vibrations

Abstract

Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and C-H stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05 mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with -turns and unordered motifs at the expense of intermolecular -sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research.

A cost-effective polyethylene glycol-based method for the isolation of functional edible nanoparticles from ginger rhizomes

“…Recently, Attenuated total reflection Fourier-transform Infrared Spectroscopy (ATR-FTIR) have been reported for simultaneous determination of protein and lipids from extracellular vesicles, without the need for extensive sample processing 28 . The distinctive absorption bands of proteins and lipid in the FTIR spectrum will www.nature.com/scientificreports www.nature.com/scientificreports/ also aid in the evaluation of Protein to Lipid (P/L) ratio.…”

“…Bruker Tensor-II series FTIR spectrometer. For protein to lipid ratio calculation, we adopted the methodology described earlier by Mihaly et al 28 10 µl of sample was mounted on the diamond ATR crystal and waited for ~45 minutes to form the thin dry film. The measurements were performed in ambient condition, immediately after thin film formation.…”

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

“…Quantification of total lipids can be achieved, e.g. by sulfophosphovanilin assay [186], by measuring fluorescence of phospholipid dyes that fluoresce only when incorporated into lipid bilayers, such as DiR [187], or by total reflection Fourier-transform infrared spectroscopy [188]. However, the latter requires specialized equipment, and the former two types of assays may be insufficiently sensitive for small amount of EVs.…”

“…Importantly, ratios of the different quantification methods may provide useful measures of purity. For example, protein:particle ratio [198,199], protein: lipid ratio [186,188,200] and RNA:particle [201] have been proposed as possible purity metrics, although their applicability across protein, lipid, RNA and particle quantification methods remains to be established. Techniques that measure multiple parameters at once, such as colloidal nanoplasmonic assays or infrared (IR) spectroscopy [188,199] may be good optional methods, despite the need for specific sensors or other equipment.…”

Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane‐Associated Bio‐macromolecules Studied with Polarized Light

“…Consequently, additional techniques that could rapidly provide further structural information on these systems are highly desirable . In this respect, we recently demonstrated that attenuated total reflection Fourier‐transform infrared (ATR‐FTIR) spectroscopy is a fast and reliable method that can provide insight into compositional changes of EV subpopulations without complicated sample preparations . To progress further towards rapid investigation of EVs at the molecular level, here we employed polarized light spectroscopy techniques, namely, linear dichroism (LD) and also circular dichroism (CD), that are commonly applied for model membrane systems, but their use in EV research is scarce.…”