Raman Spectroscopy for Protein Analysis

Abstract

Proteins are essential parts of organisms and participate in virtually every process within cells. Raman spectroscopy can be a powerful tool for the characterization of modified amino acids and proteins. In addition to the potential for quantitative results, it offers the advantage of not requiring any sample preparation. This review focuses on applications for protein analysis, published in the period 2010–2014.     

Metal‐assisted regioselectivity in nucleophilic substitutions: a study by Raman spectroscopy and density functional theory calculations

A series of complexes (Fe^II, Cu^II and Ni^II) of the N,O bidentate ligand 6,7‐dichloroquinoline‐5,8‐dione in water was investigated by using Raman spectroscopy, and the experimental peaks were assigned with the help of computed spectra by density functional theory (DFT) calculations. A strong shift to lower wavenumbers was observed for the vibration of the CO group involved in chelation, depending on the type of metal ion. When each complex was used in the substitution reaction by the nucleophilic reagent piperidine, two products having the same molecular composition but showing the substituent in different regions of the molecule were obtained, and moreover their regioselective formation was in agreement with the size of the Raman shifts previously observed for the complexes. This example confirms the potential of the approach involving Raman spectroscopy combined with DFT calculations in the characterization of metal complexes as key intermediates in organic reactions, with the possibility of predicting the metal system capable to achieve the highest selectivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Chromatographic paper embedded with silver nanostructure as a disposable substrate for surface‐enhanced Raman spectroscopy and catalytic reactor

The high cost of regular diagnostic kits severely impeded its uses for routine clinical assay and fieldworks. A cost‐effective chromatography paper is chemically modified with Ag nanostructures using the simple electroless silver deposition, producing a scalable and disposable substrate for surface‐enhanced Raman spectroscopy, as well as a large scale of catalytic active sites over many chemical reactions. Synergetic measurement including surface‐enhanced Raman spectroscopy and laser desorption ionization‐mass spectrometry is performed on Ag decorated filter paper using a thiol containing compound as indicator, allowing for the acquisition of spatially correlated spectroscopy in the tandem mode. In addition, hydrophilic porous cellulose network that contains a certain amount of liquid naturally served as a chemical reactor for molecular transport and reaction. Positive results from catalytic reaction on metallized paper convincingly demonstrated that total microanalysis system on paper (μ‐TASoP), as a compelling alternative would find a wide breadth of applications in developing disposable medical devices and customary laboratory assays. Copyright © 2015 John Wiley & Sons, Ltd.     

Raman scattering investigation of selenomethionine replacement in protein SOUL crystals

The vibrational properties of both wild‐type and selenomethionine (SeMet)‐substituted protein SOUL crystals have been investigated here by Raman spectroscopy. Several Raman peaks observed in the spectra of methionine and SeMet were identified as specific markers. The unambiguous assignment of these peaks has been inferred by comparing the experimental Raman spectra of the pure amino acids, recorded in solid state and in aqueous solution, and the Raman intensities computed using quantum chemical calculations. Moreover, a quantitative evaluation of the relative amount of SeMet replacement in the crystals of protein SOUL labelled with SeMet has been estimated through the ratio between the Raman intensities of marker peaks. These results offer evidence of the potential of Raman microscopy as a reliable and non‐invasive tool for novel in‐depth structural investigations in biocrystallography. Copyright © 2009 John Wiley & Sons, Ltd.     

芦荟中芦荟苷的结构表征及光谱分析

芦荟以其丰富的多糖、蛋白质、氨基酸、维生素、活性酶及对人体有益的微量元素被广泛用于各个领域。然而其主要活性成分芦荟苷同时也是一种致敏成分,更有甚者可能会引起严重的过敏反应。将红外、拉曼光谱应用于芦荟苷结构的表征,并将密度泛函数理论应用到理论计算中,利用B3LYP/6-31G(d)基组分析振动,可以更好地理解芦荟苷分子的振动频率。通过比较溶液的酸碱性以及最佳溶剂,最终选定在中性条件下,水作为溶剂为最佳实验条件,测定芦荟苷的检测限可以达到5 ppm的水平,为芦荟苷含量的快速检测奠定了理论基础。     

Effect of Al content on the structure of Al‐substituted goethite: a micro‐Raman spectroscopic study

The characterization of X‐ray diffraction, X‐ray fluorescence, and field emission scanning electron microscope were used to confirm the successful preparation of Al‐substituted goethite with different Al content. The micro‐Raman spectroscopy was utilized to investigate the effect of Al content on the goethite lattice. The results show that all the feature bands of goethite shifted to high wavenumbers after the occurrence of Al substitution for Fe in the structure of goethite. The shift of wavenumber shows a good linear relationship as a function of increasing Al content especially for the band at 299 cm⁻¹ (R² = 0.9992). The in situ Raman spectroscopy of thermally treated goethite indicated that the Al substitution not only hinders the transformation of goethite, but also retarded the crystallization of thermally formed hematite. All the results indicated that Raman spectrum displayed an excellent performance in characterizing Al‐substituted goethite, which implied the promising application in other substituted metal oxides or hydroxides. Copyright © 2013 John Wiley & Sons, Ltd.     

Near infrared Raman spectroscopic characterization of blood plasma of normal,oral premalignant and malignant conditions—a pilot study

The metabolic end products from cells/tissues that are released into the circulating blood stream and any changes in their level because of pathological conditions may be used as markers in disease diagnosis. Raman spectroscopy has been exploited to characterize the biomolecules present in the blood plasma of clinically confirmed normal group, premalignant (Oral Sub Mucous Fibrosis) and malignant (Oral Squamous Cell Carcinoma) at 784.15 nm. Raman spectral signatures show relatively less intense Raman bands of phenylalanine, lipid and antioxidant beta carotene but higher intense bands for proteins, DNA base components and amino acids (tyrosine and tryptophan) for malignant group than that of normal group. However premalignant group possess high intense Raman bands for amino acids (tyrosine and tryptophan) at 830, 1020 and 1620 cm⁻¹ and protein peaks at 913, 978 and 1646 cm⁻¹ when compared to that of malignant and normal group. Principal component analysis coupled with linear discriminant analysis (PCA‐LDA) yielded a diagnostic sensitivity of 96.3% and 91.2%, and a specificity of 80.0% and 96.7% in the classification of normal from premalignant and normal from malignant, respectively. This indicates that Raman spectroscopy of blood plasma has the potential in classifying normal and oral malignancy conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of Raman spectroscopy for prediction of antitumor response to silibinin and its nanoparticulates in DMBA‐induced oral carcinogenesis

Raman spectroscopy is a vibrational spectroscopic technique that can be used to monitor the therapeutic efficacy of anticancer drugs during carcinogenesis in a non‐invasive and label‐free manner. The present study aims to investigate the biochemical changes exerted upon free silibinin (SIL) and its nanoparticulate (SILNPs) treatment against 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced oral carcinogenesis in the fingerprint region of 1800–500 cm⁻¹ using HE‐785 Raman spectrometer. Raman spectra differed significantly between the control and tumor tissues, with tumor tissues characterized by increased intensities of vibrational bands such as nucleic acids, phenylalanine and tryptophan and a lower percentage of lipids when compared to the control tissues. Further, oral administration of free SIL and SILNPs significantly increased lipids and decreased the levels of tryptophan, phenylalanine and nucleic acid contents. Overall, the treatment of nanoparticulate SIL was found to be a more potent antitumor effect than free SIL in preventing the formation of tumor and also brought back the several Raman bands to a normal range in the buccal mucosa of hamsters during DMBA‐induced oral carcinogenesis. In addition, the detailed secondary structure of proteins in the control and experimental groups is also presented. Furthermore, the diagnostic algorithms based on principal component linear discriminant analysis (PC‐LDA) achieved an overall sensitivity of 94–100% and specificity of 76–100%. These results further demonstrate that Raman spectroscopy associated with PC‐LDA diagnostic algorithms could be a valuable tool for rapid and sensitive detection of specific biomolecular changes at the molecular level in response to anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of biologically active diterpenoic acids by Raman Spectroscopy

Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional Raman spectroscopy is not suitable for their unambiguous identification, especially not in solution. We attempted to increase the sensitivity by applying UV‐resonance Raman spectroscopy and surface‐enhanced Raman spectroscopy (SERS) techniques. The UV‐Raman spectra of the three compounds in ethanol/water 50:50 showed only very few enhanced Raman lines. SERS spectra with 514‐nm excitation with Ag colloids were also relatively weak. The best SERS spectra were obtained with 785‐nm excitation on a novel nanostructured substrate, ‘black silicon’ coated with a 400‐nm gold layer. The spectra showed clear differences, and these ‘fingerprints’ would be suitable for the unambiguous identification of these diterpenoic acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Effective methods for preparation and characterization of HNO‐adducts of heme proteins

Driven by the documented effectiveness of cryoradiolysis coupled with resonance Raman spectroscopy for the preparation and structural characterization of unstable peroxo and hydroperoxo intermediates of catalytic and oxidative enzymes, similar strategies have now been applied to prepare and study the still relatively unexplored chemistry of nitrosyl hydride (HNO) adducts of heme proteins and their derivatives. Previously, such HNO derivatives of heme proteins were prepared by chemical addition of reducing agents or by direct addition of an HNO donor. Here, for the first time, we report effective cryoradiolytic preparation of Fe–N(H)O species of myoglobin and their structural characterization by resonance Raman spectroscopy. Our results are in excellent agreement with those previously reported for chemically prepared derivatives and with computational results. Furthermore, the present study provides new data for the deuterated analogue; i.e. bearing a nitrosyl deuteride (DNO) fragment. Copyright © 2012 John Wiley & Sons, Ltd.     

Raman spectroscopic analysis of cataract lens: A compendious review

Age-related cataracts is a pressing health issue with the increase in elderly populations. This creates an imminent demand for the development of an early, noninvasive method of cataracts diagnosis. Early detection of cataracts will improve quality of life and may prevent morbidity associated with advanced cataracts and surgery. Raman spectrum of proteins provides characteristic information regarding molecular interactions of peptide residues. Hence Raman spectroscopy is a promising tool for the study of protein-related diseases, such as cataracts. We surveyed the literature to assess the use of Raman technology in the studies of human lens and animal models. These studies included analysis of amino acids (i.e., cysteine, tryptophan, and tyrosine, etc.) and secondary protein structures (i.e., α-helix and β-sheet) in various Raman profiles. Other studies used Raman spectroscopy to analyze and monitor the development of cataracts in lens. Technological advances in the instrumentation of laser Raman spectroscopy, including Fourier transform Raman spectroscopy, Raman microspectroscopy, and confocal Raman microspectroscopy have improved the performance of Raman spectroscopic analysis. How to take advantage of these developments and make it closer to reality using Raman spectroscopic methods to diagnose cataracts in a timely manner is a key challenge for the scientific community of Raman spectroscopy.     

Solution-phase reductive cyclization of 2-quinoxalinol analogs: Systematic study of parallel synthesis

1,5-Difluoro-2,4-dinitrobenzene starting material was treated via primary and/or secondary substitution with a variety of amino acids or amines and the aromatic m-dinitro groups were then reductively cyclized provide the 2-quinoxalinol analogs. The conditions for 1,5-dialkylamino-2,4-dinitrobenzene reduction have been systematically studied and optimized in solution. Three effective methods are described for the high-throughout generation of 2-quinoxalinol analogs.     

Near‐infrared surface‐enhanced Raman spectroscopy (NIR‐SERS) studies on oxyheamoglobin (OxyHb) of liver cancer based on PVA‐Ag nanofilm

A near‐infrared surface‐enhanced Raman spectroscopy (NIR‐SERS) method was employed for oxyheamoglobin (OxyHb) detection to develop a simple blood test for liver cancer detection. Polyvinyl alcohol protected silver nanofilm (PVA‐Ag nanofilm) used as the NIR‐SERS active substrate to enhance the Raman scattering signals of OxyHb. High quality NIR‐SERS spectrum from OxyHb adsorbed on PVA‐Ag nanofilm can be obtained within 16 s using a portable Raman spectrometer. NIR‐SERS measurements were performed on OxyHb samples of healthy volunteers (control subjects, n = 30), patients (n = 40) with confirmed liver cancer (stage I, II and III) and the liver cancer patients after surgery (n = 30). Meanwhile, the tentative assignments of the Raman bands in the measured NIR‐SERS spectra were performed, and the results suggested cancer specific changes on molecule level, including a decrease in the relative concentrations and the percentage of aromatic amino acids of OxyHb, changes of the vibration modes of the C_aH_m group and pyrrole ring of OxyHb of liver cancer patients. In this paper, principal component analysis (PCA) combined with independent sample T test analysis of the measured NIR‐SERS spectra separated the spectral features of the two groups into two distinct clusters with the sensitivity of 95.0% and the specificity of 85.7%. Meanwhile, the recovery situations of the liver cancer patients after surgery were also assessed using the method of discriminant analysis‐predicting group membership based on PCA. The results show that 26.7% surgeried liver cancer patients were distinguished as the normal subjects and 63.3% were distinguished into the cancer. Our study demonstrated great potentials for developing NIR‐SERS OxyHb analysis into a novel clinical tool for non‐invasive detection of liver cancers. Copyright © 2013 John Wiley & Sons, Ltd.     

Microwave-enhanced liquid-phase synthesis of thiohydantoins and thioxotetrahydropyrimidinones

An efficient, microwave-assisted method for the liquid-phase combinatorial synthesis of 3,5-disubstituted thiohydantoins and 3,5-disubstituted 2-thioxotetrahydropyrimidin-4-ones has been developed. In synthesizing thiohydantoins, Fmoc-protected amino acids were coupled with polymer support and then deprotected to give primary amines. While in synthesizing thioxotetrahydropyrimidinones, 3-chloropropionyl chloride was immobilized to the support and subsequently reacted with various amines to form secondary amines. The PEG bound primary/secondary amines then were incorporated with various isothiocyanates to give thiourea intermediates and concomitant cyclization/cleavage steps occurred under mild basic condition. The desired products were then liberated from the soluble matrix in good yield and purity. All reactions described here were performed under microwave irradiation.     

Acetic acid effects on enhancement of growth rate and reduction of amorphous carbon deposition on CNT arrays along a growth window in a floating catalyst reactor

The mm-long carbon nanotube (CNT) arrays were grown in a floating catalyst reactor, using xylene-ferrocene and a small amount of acetic acid as the feed. The CNT arrays deposited on a quartz substrate at several positions along the reactor were extensively characterized using Raman spectroscopy, scanning electron microscopy, X-ray diffraction, high-resolution transmission electron microscopy, and optical microscopy. Various characterization methods consistently reveal that the acetic acid additive to the feed alleviates deposition of amorphous carbon layer, which gradually thickens CNTs along the reactor. The acetic acid also resulted in a higher growth rate along the so-called growth window, where CNT arrays are deposited on the quartz substrate. High-performance liquid chromatography of extracted byproducts (PAHs) confirmed the presence of some polycyclic aromatic hydrocarbons. The solid weight of PAHs decreased upon addition of ferrocene as the catalyst precursor, as well as of acetic acid to xylene feed. The results suggest that primary light products of xylene pyrolysis can be competitive reactants for both catalytic and subsequent pyrolytic reactions. They may also be more efficient feeds for CNT growth than xylene itself.     

EPR spectroscopic characterization of biological thiyl radicals as PBN spin-trap adducts

While the importance of thiols and their derivatives in biological processes is widely appreciated, the elucidation of the roles played by thiyl radicals in these processes — being hampered by the radical reactivity that makes their detection and characterization difficult — is lagging. The results of a spin-trap EPR study are reported which advance the capability for detecting and identifying thiyl radicals. Adducts with PBN (α-phenyl-N-_t-butylnitrone) of thiyl radicals derived from the biologically abundant low-molecular-weight thiols cysteine, homocysteine, and glutathione are examined. Significant differences in the β-proton hyperfine couplings of the various adducts are observed; both the EPR lineshapes and the radical adduct lifetimes show trends reflective of the molecular size of the trapped thiyl radical. These results indicate that EPR spectroscopy can be useful in identifying specific thiyl radicals that may be involved in the biochemical reactions of low-molecular-weight thiols, protein thiols, and their derivatives.     

Raman spectroscopy of carbonaceous particles of environmental interest

Carbonaceous particles of environmental origin are practically insoluble, which constitutes an obstacle to many chemical physical characterization techniques. Micro‐Raman spectroscopy overcomes the sample preparation step and can be applied on the as‐received sample and even in situ, with nowadays commercially available portable Raman equipment. Multivariate statistical analysis of Raman data recorded on a variety of carbonaceous particles of different origin, through a molecular interpretation of the signal, shows that the use of Raman spectroscopy with several excitation sources can provide a useful tool for assisting the characterization of various forms of environmental carbon matter. Copyright © 2015 John Wiley & Sons, Ltd.     

Outdoor use of mobile Raman spectrometers to study the solutions and ices of amino acids

Selected amino acids (glycine, alanine, and threonine) were analysed in the form of water solutions, mixtures of these amino acids in solution, as well as their respective ices using two hand‐held instruments under outdoor and winter mountain conditions. The hand‐held instruments featured 785 and 532 nm excitations; the latter being one of the first using this wavelength on the market. The changes to the Raman spectra of the solutions and ices are discussed in relationship to the degree of ambiguity of the detection of the amino acids within the mixtures. The hand‐held instruments were able to detect two amino acids out of the three in solution. The results showed that the hand‐held instruments provide sufficient quality spectra when analysing ices prepared from the solutions; however, an unambiguous detection of these amino acids within mixtures still remains a challenge. Copyright © 2014 John Wiley & Sons, Ltd.     

Raman scattering features of lead pyroantimonate compounds: implication for the non‐invasive identification of yellow pigments on ancient ceramics. Part II. In situ characterisation of Renaissance plates by portable micro‐Raman and XRF studies

The effectiveness of Raman spectroscopy (using a bench‐top system on standard pigments) for the characterisation of modified lead antimonate yellows was demonstrated in the already published Part I. The knowledge gained is employed here for the study of yellow glazes on genuine Renaissance plates with the aim of identifying non‐invasively lead pyroantimonate compounds by portable micro‐Raman equipment. The investigation was carried out directly on site, at the Victoria and Albert Museum (London, UK) and at the Museo Statale d'Arte Medievale di Arezzo (Arezzo, Italy), combining Raman and X‐ray fluorescence analyses. In addition to the spectral features of both unmodified Naples yellow and Sn‐ and Zn‐modified lead antimonate compounds, the Raman patterns related to partially modified pyrochlore structures were observed. For this reason, the possible Sn‐induced modification of Naples yellow by cassiterite (SnO₂), present within the glaze as opacifier was explored on lead antimonate yellow glaze mock‐ups fired at different temperatures. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of chemical modification in single human keratinocyte cells exposed to low doses of chlorpyriphos by Raman micro‐spectroscopy

Raman micro‐spectroscopy can be used to investigate biological single cells exposed to different chemicals. Since chronic exposure at low doses of pesticides can promote several diseases, the investigation of cellular changes induced by exposure to non‐cytotoxic doses of pesticides is of increasing interest. The efficiency of Raman micro‐spectroscopy to detect chemical modification in normal human keratinocytes induced by exposure to non‐cytotoxic doses of chlorpyriphos, an organophosphate pesticide present in many plant‐protection products, was investigated. Such modification affects mainly proteineous components (both single amino acids and amide linkages between amino acids) of the nucleus, cellular membranes and cytoplasm as well as the nucleic acid component of the nucleus. Chemical modifications are already detectable after 24 h exposure of keratinocytes at a chlorpyriphos concentration of 10⁻⁶ M , which is three orders of magnitude lower than the cytotoxic concentration (10⁻³ M ). Heavy damage to the lipid component occurs after exposure to the nearly cytotoxic concentration (10⁻⁴ M ). Atomic force microscopy images of keratinocyte cells exposed for 24 h to various chlorpyriphos concentrations show a progressive deterioration of the morphology of cellular membrane as the chlorpyriphos concentration increases. The results of this work may have wide applications in the monitoring of molecular changes in single human cells exposed to toxic agents. Copyright © 2010 John Wiley & Sons, Ltd.