Structural Characterization of Membrane Proteins and Peptides by FTIR and ATR-FTIR Spectroscopy

Exosomes are nanosized vesicles containing specific cargos of DNA, RNA, proteins, metabolites, and intracellular and membrane lipids. Exosome isolation needs to be optimized carefully depending on the type of biofluid and tissue and the retrieved exosomes need to be characterized. The main objective of this study was to determine the feasibility of a multimodal analysis of Attenuated Total Reflectance – Fourier Transform Infrared (ATR-FTIR) spectroscopy and UPLC–QqTOF-MSMS for the development of a routine quality control tool of isolated exosomes and the rapid characterization of their lipid profiles and total protein content. Using human milk as model example, exosomes were isolated by multi-stage ultracentrifugation. After single-phase extraction, lipidomic analysis was carried out by UPLC–QqTOF-MSMS with automated MSMS-based annotation using HMDB, METLIN, LipidBlast and MSDIAL databases. The classes with the largest number of annotated features were glycerophospholipids, sphingolipids, and glycerolipids. Then, dry films of 2 ​μL exosomes were directly analyzed by ATR-FTIR. Multivariate analysis showed significant associations between ATR-FTIR specific regions and the concentrations of different lipid classes. Principal component analysis and Hierarchical Cluster Analysis of IR and lipidomic data showed that ATR−FTIR renders valuable qualitative descriptors of the lipid content of isolated exosomes. Total LC-MS lipid and total protein contents could also be quantified by using the area of CHs and CO stretching bands as well as the amide I band. As a conclusion, results obtained show that multimodal analysis of ATR-FTIR and UPLC-MS data is a useful tool for the development of spectroscopic methods. ATR-FTIR provided both, qualitative and quantitative chemical descriptors of isolated exosomes, enabling a fast and direct quantification of total protein and lipid contents.

Introduction: Autistic Spectrum Disorder (ASD) is a neurodevelopmental syndrome diagnosed by clinical observations and behavioral assessments. Currently, diagnostic methods based on biomarkers are lacking, making the development of accurate and appropriate diagnostic tools for ASD a challenge. The reduction in waiting time for the diagnosis of ASD can anticipate the beginning of interventions, providing better results. Therefore, the search for an economic, non-invasive, sustainable (without reagents) and accurate diagnostic platform is relevant to the diagnosis of ASD. Objective: Establish salivary infrared spectral signature in the TEA that allows the development of diagnostic platforms based on Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy system. Material and methods: This study was approved by the Research Ethics Committee (CEP) in human beings at the Federal University of Uberlândia (UFU) and analyzed the salivary profile of 53 participants, 19 diagnosed with ASD and 34 neurotypical (NT), with ages between 2 and 20 years, using ATR-FTIR spectroscopy in regions with an electromagnetic spectrum between 4000 cm-1 and 400 cm-1. The sensitivity and specificity of the vibrational modes were evaluated using the Receiver Operating Characteristic (ROC) curve. Results: Three salivary vibrational modes in the ASD group were differentially expressed (p <0.05) compared to NT. These vibrational modes have been pre-validated as potential spectral diagnostic biomarkers by analyzing the ROC curve. The band area of the 2935 vibrational mode (2942 - 2895 cm-1) showed a sensitivity of 73.68% and specificity of 79.41% (p = 0.012) compared with the NT, and accuracy of 76.5%. The areas of the band in the 1636 vibrational mode (1738 - 1581 cm-1) of the ASD group showed a sensitivity of 68.42% and specificity of 73.53% (p = 0.0032) compared to the NT area, and of the vibrational mode 986 ( 1005 - 959 cm-1) exhibited a sensitivity of 50% and specificity of 73.68% (p = 0.0493). Conclusion: The salivary spectral biomarkers discovered using univariate analysis can support a new robust alternative for the complementary diagnosis of ASD from a sustainable technology platform, with an ultrasmall sample volume, non-invasive collection and without the use of reagents, which contributes for making medical decisions and favors the beginning of the indicated intervention. Keywords: Autism spectrum disorder. Saliva. Biomarkers. Fourier transform infrared spectroscopy. Diagnosis.

INTRODUCTION: The ability to diagnose brain tumours rapidly from serum would allow for rapid testing and decreased time to results providing a responsive diagnostic environment. ATR-FTIR (Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy) generates a serum sample - fingerprint - since biomolecules exhibit different responses to different wavelengths of light. We have previously used ATR-FTIR to discriminate high grade brain cancer, low-grade brain cancer and normal serum to sensitivities and specificities on average of 93.75 and 96.53 % respectively. This paper reports the analysis of 429 patients. METHOD: 1 µl of serum was analysed in triplicate from 429 patients via ATR-FTIR. This dataset was analysed using pattern recognition algorithms for the following models 1) Cancer (n = 314) vs Non-cancer (n = 122), 2) Metastatic Brain Cancer (n = 192) vs Brain Cancer (n = 124) vs Non-Cancer (n = 122), 3) Organ of tumour origin, 4)High grade glioma (n = 52) vs low grade glioma (n = 24) vs meningioma (n = 47) 5) Subtype of Brain Cancer. RESULTS: ATR-FTIR spectroscopy was able to discriminate different groupings with high accuracy. The predicted range for each diagnostic layer was between 78.26 - 100.00 % sensitivity and specificity respectively. Interrogation of the loadings plots showed differences in Amide and lipid content of the serum. CONCLUSION: A robust reproducible method has been developed for the diagnosis of small volumes of serum. In addition multiple layers of diagnostic information can be provided from the same spectral dataset. ATR-FTIR has shown to be an excellent clinical tool for rapid diagnosis enabling a dynamic and responsive clinical environment. Future work will investigate spectroscopic changes during treatment.

In order to improve the gas barrier characteristics of PET (polyethylene terephthalate) we have carried out two experiments; a preliminary experiment using film samples and an applied experiment to modifying inner surfaces of bottles. The measurements of film samples by both ATR FT-IR (Attenuated Total Reflection Fourier Transform Infrared) spectroscopy and Laser Raman spectroscopy show that most PET molecules in the near surface volume are destroyed and amorphized by ion implantation. Gas permeability of the sample is also measured. For applying PBII (Plasma Based Ion Implantation) to bottles we develop a new technique to implant ions onto the inner surfaces while plasma is generated inside the bottle. An electrode that is supplied with positive high-voltage pulses differently from negative ones as in the preliminary experiment is inserted into the bottle. The inner surface of the PET bottle is investigated by both ATR FT-IR spectroscopy and Laser Raman spectroscopy, too.

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.

The composition estimate of LDPE/HDPE blends is still a challenge for the most polymer characterization techniques due to partial miscibility effects and similar chemical structures between the polymeric components. Herein, we report the usage of Attenuated Total Reflectance - Fourier Transform Infrared (ATR-FTIR) spectroscopy and Competitive Adaptive Reweighted Sampling - Partial Least Squares (CARS-PLS) linear regression as a powerful tool to determine the content of LDPE/HDPE blends. Several regression parameters (pretreatment method, Monte Carlo sampling runs, k-fold and maximal number of latent variables for cross-validation) were analyzed for this new ATR-FTIR application. Although LDPE and HDPE have approximate FTIR spectra, the CARS-PLS-based models show good prediction performance for the whole composition range of HDPE/LDPE blends, with prediction errors in the range of 4.9–6.58 wt% for HDPE amount.

This study investigated the physico-chemical properties of the binders used in ancient Chinese wall paintings under repeated freeze–thaw cycles. Gum Arabic, cow bone, and fish bladder glue samples were prepared before and after three, six, and nine freeze–thaw cycles. The surface morphology, crystallinity, and spectral characteristics were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structures of the protein binders were also analyzed before and after thaw–freeze cycles. The results show that the surface morphologies of the gum Arabic, cow bone, and fish bladder glue gradually changed from undulating to smooth as the number of freeze–thaw cycles increased. The intensities of the XRD peaks and crystallinity of the binders gradually decreased after six freeze–thaw cycles. ATR-FTIR spectroscopy indicated that no significant changes occurred in the original chemical compositions of the binders after multiple freeze–thaw cycles. The wavenumbers of the characteristic peaks remained unchanged but the intensities of the peaks changed significantly. Analysis of the secondary protein structures indicated that the α-helix contents of the cow bone and fish bladder glue samples decreased significantly, whereas the parallel β-sheet and random coil contents increased.

Machine learning-enhanced Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy for discrimination of green Arabica and Robusta coffee beans

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

Phospholipid-protein balance in affective disorders: Analysis of human blood serum using Raman and FTIR spectroscopy. A pilot study

Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) spectroscopy is a label-free, non-destructive analytical technique that can be used extensively to study a wide variety of molecules in different conditions. Proteins have very complicated three dimensional structures with multi-level conformations, which are highly correlated with their biological activities. Recently, there is a significant increase of materials based on interaction between proteins and nanoparticles. The aim of this paper is to highlight the understanding of protein interaction with silver nanoparticles (AgNPs) surfaces. Information about the secondary structures of collagen with and without AgNPs was obtained from atomic force microscopy (AFM) measurements.ATR-FTIR spectroscopy was used for monitoring the changes in the secondary structures of collagen upon interaction with AgNPs. Amide I is the most sensitive band for detecting changes in the protein secondary structures. Its characteristic absorption band is located at 1600–1700 cm-1. Comparing the spectra of collagen with and without AgNPs in this region, information about the different types of secondary structures such as α-helix, β-sheets, turns and random coil can be obtained. The conjugation of AgNPs-collagen occurred mainly through electrostatic interactions. Based on these data, the effects of AgNPs stability and the conformational changes of collagen upon interaction with the AgNPs are discussed.

Surface discharge inception voltage (SDIV) in copper sulphide diffused Oil Impregnated Pressboard (OIP) material reduces with increases in harmonic content in the supply voltage, irrespective of Total Harmonic Distortion (THD). The rise time of current signal injected during Surface discharge process is about few ns and the Fast Fourier transform (FFT) analysis of Ultra High Frequency (UHF) signal formed due to surface discharge process have dominant frequency of 0.9 GHz. The characteristics are the same irrespective of level of thermal aging of OIP and amount of DBDS present in transformer oil. The amplitude and width of the current pulse increases with increases due to aging. Atomic Absorption Spectroscopy (AAS) studies results indicate that the amount of copper content in pressboard material have increased with increasing in concentration level of DBDS. Energy dispersive spectroscopy (EDS) conform the amount of copper and sulphur is high with thermally aged OIP due to aging of OIP in transformer oil with DBDS concentration. Attenuated Total Reflectance - Fourier Transform Infrared (ATR-FTIR) spectroscopy studies indicates the formation of aldehydes and carboxylic acids, caused due to degradation of cellulose structure. Phase Resolved Partial Discharge (PRPD) analysis indicates that the surface discharge activity occurs near the peak of the applied AC/harmonic AC voltages at inception voltage and at rising portion of the applied AC/harmonic AC voltage at higher voltages. The tensile and flexural strength of the material reduces with thermal aging of OIP material, irrespective of concentration of DBDS in oil.

Rapid classification of chromoblastomycosis agents genera by infrared spectroscopy and chemometrics supervised by sequencing of rDNA regions

Analytical applications of Fourier transform-infrared (FT-IR) spectroscopy in microbiology and prion research

Abstract. Retrieving high-precision concentrations of atmospheric trace gases from FTIR (Fourier transform infrared) spectrometry requires a precise knowledge of the instrumental performance. In this context, this paper examines the impact on the ozone (O3) retrievals of several approaches used to characterize the instrumental line shape (ILS) function of ground-based FTIR spectrometers within NDACC (Network for the Detection of Atmospheric Composition Change). The analysis has been carried out at the subtropical Izaña Observatory (IZO, Spain) by using the 20-year time series of the high-resolution FTIR solar absorption spectra acquired between 1999 and 2018. The theoretical quality assessment and the comparison to independent O3 observations available at IZO (Brewer O3 total columns and electrochemical concentration cell, ECC, sondes) reveal consistent findings. The inclusion of a simultaneous retrieval of the ILS parameters in the O3 retrieval strategy allows, on the one hand, a rough instrumental characterization to be obtained and, on the other hand, the precision of the FTIR O3 products to be slightly improved. The improvement is of special relevance above the lower stratosphere, where the cross-interference between the O3 vertical distribution and the instrumental performance is more significant. However, it has been found that the simultaneous ILS retrieval leads to a misinterpretation of the O3 variations on daily and seasonal scales. Therefore, in order to ensure the independence of the O3 retrievals and the instrumental response, the optimal approach to deal with the FTIR instrumental characterization is found to be the continuous monitoring of the ILS function by means of independent observations, such as gas cell measurements.