Raman spectroscopy and imaging: applications in human breast cancer diagnosis

The applications of spectroscopic methods in cancer detection open new possibilities in early stage diagnostics. Raman spectroscopy and Raman imaging represent novel and rapidly developing tools in cancer diagnosis. In the study described in this paper Raman spectroscopy has been employed to examine noncancerous and cancerous human breast tissues of the same patient. The most significant differences between noncancerous and cancerous tissues were found in regions characteristic for the vibrations of carotenoids, lipids and proteins. Particular attention was paid to the role played by unsaturated fatty acids in the differentiation between the noncancerous and the cancerous tissues. Comparison of Raman spectra of the noncancerous and the cancerous tissues with the spectra of oleic, linoleic, α-linolenic, γ-linolenic, docosahexaenoic and eicosapentaenoic acids has been presented. The role of sample preparation in the determination of cancer markers is also discussed in this study.     

Characterization of lipid extracts from brain tissue and tumors using Raman spectroscopy and mass spectrometry

Brain tissue is characterized by high lipid content. The amount of lipids decreases, and its composition changes in the most frequent primary brain tumor, the glioma. Scope of the current paper was to extract quantitatively lipids from porcine and human brain tissue as well as from five human gliomas using a modified protocol according to Folch. The lipid extracts were studied by Raman spectroscopy with 785 nm excitation and by mass spectrometry with electron impact ionization. Porcine and human brain tissues have similar water and lipid content and show similar Raman and mass spectra. In contrast, gliomas are characterized by increased water content and decreased lipid content. Elevated phosphatidylcholine to cholesterol ratios in lipid extracts of gliomas were indicated by Raman bands of the choline group and cholesterol. Due to its higher sensitivity, mass spectrometry detected increased levels of cholesterol ester relative to cholesterol in lipid extracts of gliomas. For comparison, thin tissue sections were prepared from the glioma specimens before lipid extraction; infrared spectroscopic images were recorded and analyzed by a supervised classification model. This study demonstrates how to improve the analysis of brain tumors and to complement the diagnosis of brain pathologies using a multimodal approach.     

Probing the interaction between prostacyclin synthase and prostaglandin H2 analogues or inhibitors via a combination of resonance Raman spectroscopy and molecular dynamics simulation approaches

In an aim to probe the structure-function relationship of prostacyclin synthase (PGIS), resonance Raman (RR) spectroscopy and molecular dynamic (MD) simulation approaches have been exploited to characterize the heme conformation and heme-protein matrix interactions for human PGIS (hPGIS) and zebrafish PGIS (zPGIS) in the presence and absence of ligands. The high-frequency RR (1300-1700 cm(-1)) indicates that the heme group is in the ferric, six-coordinate, low-spin state for both resting and ligand-bound hPGIS/zPGIS. The low-frequency RR (300-500 cm(-1)) and MD simulation reveal a salient difference in propionate-protein matrix interactions between hPGIS and zPGIS, as evident by a predominant propionate bending vibration at 386 cm(-1) in resting hPGIS, but two vibrations near 370 and 387 cm(-1) in resting zPGIS. Upon binding of a substrate analogue (U46619, U51605, or U44069), both hPGIS and zPGIS induce a distinctive perturbation of the propionate-protein matrix interactions, resulting in similar Raman shifts to ~381 cm(-1). On the contrary, the bending vibration remains unchanged upon binding of inhibitor/ligand (minoxidil, clotrimazole, or miconazole), indicating that these inhibitors/ligands do not interfere with the propionate-protein matrix interactions. These results, together with subtle changes in vinyl bending modes, demonstrate drastically different RR shifts with heme conformational changes in both hPGIS and zPGIS upon different ligand bindings, suggesting that PGIS exhibits a ligand-specific heme conformational change to accommodate the substrate binding. This substrate-induced modulation of the heme conformation may confer high product fidelity upon PGIS catalysis.     

Correlation of surface-enhanced Raman spectroscopy and laser desorption-ionization mass spectrometry acquired from silver nanoparticle substrates

Applying complementary experiments, like laser desorption-ionization mass spectrometry (LDI-MS) and confocal surface-enhanced Raman microscopy, to the same physical sample location has the potential to elucidate the behavior of complex chemical and biochemical systems in ways that are not available to either method applied in isolation. In these experiments surface-enhanced Raman scattering (SERS) and LDI-MS are applied to the same sample spot using a common structure, deposited Ag colloids, both as ionization matrix and simultaneously as enhancing media for surface-enhanced Raman scattering of small organic molecules, dyes and lipids, and the behavior is compared. Three compounds-p-aminothiophenol (ATP), rhodamine 6G and cholesterol-which exhibit different strengths of interaction with Ag are examined in detail by correlated SERS and LDI-MS. The related mechanisms of nanoparticle-assisted desorption-ionization and Raman enhancement are explored by correlating mass and Raman spectra. The correlated spectra highlight the manner in which the different test compounds interact with plasmonic metal nanostructures. These coupled studies yield new insight into the transition of analyte from the metal-solution interface to gaseous ions, including, in the case of organothiols, a rich set of mixed clusters that provide chemical insight into the ion formation process.     

Reaction products in mass spectrometry elucidated with infrared spectroscopy

Determining the structure and dynamics of large biologically relevant molecules is one of the key challenges facing biology. Although X-ray crystallography (XRD) and nuclear magnetic resonance (NMR) yield accurate structural information, they are of limited use when sample quantities are low. Mass spectrometry (MS) on the other hand has been very successful in analyzing biological molecules down to atto-mole quantities and has hence begun to challenge XRD and NMR as the key technology in the life sciences. This trend has been further assisted by the development of MS techniques that yield structural information on biomolecules. Of these techniques, collision-induced dissociation (CID) and hydrogen/deuterium exchange (HDX) are among the most popular. Despite advances in applying these techniques, little direct experimental evidence had been available until recently to verify their proposed underlying reaction mechanisms. The possibility to record infrared spectra of mass-selected molecular ions has opened up a novel avenue in the structural characterization of ions and their reaction products. On account of its high pulse energies and wide wavelength tunability, the free electron laser for infrared experiments (FELIX) at FOM Rijnhuizen has been shown to be ideally suited to study trapped molecular ions with infrared photo-dissociation spectroscopy. In this paper, we review recent experiments in our laboratory on the infrared spectroscopic characterization of reaction products from CID and HDX, thereby corroborating some of the reaction mechanisms that have been proposed. In particular, it is shown that CID gives rise to linear fragment ion structures which have been proposed for some time, but also yields fully cyclical ring structures. These latter structures present a possible challenge for using tandem MS in the sequencing of peptides/proteins, as they can lead to a scrambling of the amino acid sequence information. In gas-phase HDX of an amino acid it is shown that the structure can be changed from a charge solvated to a zwitterionic structure, thereby demonstrating that HDX can be an invasive technique, in fact changing the structure of the analyte. These results emphasize that more fundamental work is required in order to understand the underlying mechanisms in two of the most important structural techniques in MS.     

Probing sites of histidine phosphorylation with iodination and tandem mass spectrometry

Phosphorylation at histidine residues occurs frequently in biology, but is often overlooked in proteomics experiments due to extreme acid lability. A new method utilizing histidine labeling with iodine to record information about phosphorylation is described. Essentially, phosphorylated histidine residues are not labeled while unmodified histidine undergoes complete iodination. Iodination is stabile both under acidic conditions, and upon collisional activation in the gas phase. This enables site-specific information to be retained with standard liquid chromatography separations and tandem mass spectrometry by collisional activation. Semi-quantitative information about the relative amounts of phosphorylated versus unmodified states can also be easily obtained from the relative ion abundances. This new method should provide a pathway forward for analyzing histidine phosphorylation in complex systems.     

Interaction of hypericin with serum albumins: surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling study

Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 A between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.     

Combination of HPLC with organic and inorganic mass spectrometry to study the metabolic response of the clam Scrobicularia plana to arsenic exposure

Arsenic is a toxic element extensively studied in the marine environment due to differential toxicological effects of inorganic and organic species. In the present work, the bivalve Scrobicularia plana was exposed to As^V (10 and 100 μg/L) for 14 days to evaluate the metabolic perturbations caused by this element. Arsenic speciation and metabolomic analysis were performed in the digestive gland of the bivalve using two complementary analytical platforms based on inorganic and organic mass spectrometry. It has been observed the greater presence of the innocuous specie arsenobetaine produced in this organism as defense mechanism against arsenic toxicity, although significant concentrations of methylated and inorganic arsenic were also present, depending on the level of arsenic in aqueous media. Complementarily, a metabolomic study based on mass spectrometry and statistical discriminant analysis allows a good classification of samples associated to low and high As(V) exposure in relation to controls. About 15 metabolites suffer significant changes of expression by the presence of As(V): amino acids, nucleotides, energy‐related metabolites, free fatty acids, phospholipids and triacylglycerides, which can be related to membrane structural and functional damage. In addition, perturbation of the methylation cycle, associated with the increase of homocysteine and methionine was observed, which enhance the methylation of toxic inorganic arsenic to less toxic dimethylarsenic.     

Enhanced Raman spectroscopy coupled to chemometrics for identification and quantification of acetylcholinesterase inhibitors

In this work, we present a new complete method using Surface Enhanced Raman Spectroscopy (SERS) and chemometrics for the qualitative and quantitative detection of pesticides by measuring the acetylcholinesterase (ACHE) activity. The Raman SERS is not only used for measuring the ACHE activity, but also for the direct detection of pesticides individually and for their identification. Gold nanoparticles (AuNPs) were used as dynamic SERS substrates for sensitive monitoring of ACHE activity in the presence of very low levels of organophosphate and carbamate pesticides, chemical warfare agents that are known to be ACHE inhibitors. The lowest detectable level for paraoxon was determined at 4.0 × 10⁻¹⁴ M and 1.9 × 10⁻⁹ M for carbaryl. The use of the enzyme allowed limits of detection for both pesticides that were much lower than the limits obtained by direct SERS analysis of the pesticides. The system shows a linear relationship between the intensity band at 639 cm⁻¹ and pesticide concentration. These results suggest that this biosensor could be used in the future for the non-selective detection of all ACHE inhibitors at very low concentrations with possible identification of the inhibitor.     

Combining surface-enhanced Raman spectroscopy and paper spray mass spectrometry for the identification and confirmation of psychotropic substances in alcoholic beverages

Criminal practices in which an individual becomes vulnerable and prone to sexual assault after ingesting drinks spiked with doping substances have become a social concern globally. As forensic protocols require a multi-tiered strategy for chemical evidentiary analysis, the backlog of evidence has become a significant problem in the community. Herein, a fast, sensible, and complementary dual analytical methodology was developed using a single commercial paper substrate for surface-enhanced Raman spectroscopy (SERS) and paper spray mass spectrometry (PS-MS) analysis to identify psychotropic substances added to alcoholic beverages irrefutably. To study and investigate this criminal practice, pharmaceutical formulations containing distinct psychotropic substances (zolpidem, clonazepam, diazepam, and ketamine) were added to drinks typically consumed at parties and festivals (Pilsen beer, açaí Catuaba®, gin tonic, and vodka mixed with Coca-Cola Zero®). A simple liquid–liquid extraction with a low-temperature partitioning (LLE-LTP) procedure was applied to the drinks and effectively minimized matrix effects. As a preliminary analysis, SERS spectra combined with Hierarchical Clustering Analysis (HCA) provided sufficient information to investigate the samples further. The presence of the protonated species for the psychotropic substances in the spiked drinks was readily verified in the mass spectra and confirmed by tandem mass spectrometry. Finally, the results demonstrate the potential of this methodology to be easily implemented into the routine of forensic laboratories and to be further employed at harm reduction tends at parties and festivals to detect contaminated beverages promptly and irrefutably as an efficient tool to prevent such crimes.     

A fully validated method for the simultaneous determination of 11 antihistamines in breast milk by gas chromatography–mass spectrometry

Antihistamines are excreted into breast milk in small amounts; however, there are no adequate published studies or data concerning their effects on newborns and safety during breastfeeding. Thus, the development of sensitive and specific methodologies for the determination of antihistamines in breast milk is critical. A simple and sensitive GC–MS method for the simultaneous determination of 11 antihistamines (diphenhydramine, orphenadrine, chlorpheniramine, dimethindene, meclozine, hydroxyzine, loratadine, desloratadine, cetirizine, rupatadine and ebastine) in breast milk was developed and validated. The antihistamines were solid‐phase extracted and derivatized with acetic anhydride and n‐propanol. Diazepam‐d₅, hydroxyzine‐d₄ and cetirizine‐d₈ were used as internal standards. Absolute recovery values for all analytes ranged from 70.5 to 120.0%, while the limits of detection and quantification for all analytes were 1.50 and 5.00 ng/mL, respectively. All calibration curves were linear (R² ≥ 0.990) within the range 5.00–1000.0 ng/mL. Accuracy (E_r) ranged between −7.6 and 7.0%, while precision (RSD) was <12% for all antihistamines. The developed method is suitable for the investigation of antihistamine‐related clinical cases, as well as for pharmacokinetic and breastfeeding safety studies.     

Probing the stability and structure of metalloporphyrin complexes with basic peptides by mass spectrometry

The stability and structure of non-covalent complexes of various peptides contatining basic amino acid residues (Arg, Lys) with metalloporphyrins were studied in a quadrupole ion trap mass spectrometer. The complexes of heme and three other metalloporphyrins with a variety of basic peptides and model systems were formed via electrospray ionization (ESI) and their stability was probed by energy-variable collision-induced dissociation (CID). A linear dependence for basic peptides and model compounds/metalloporphyrin complexes was observed in the plots of stability versus degrees of freedom and was used to evaluate relative bond strength. These results were then compared with previous data obtained for complexes of metalloporphyrins with His-containing peptides and peptides containing no basic amino acids. The binding strengths of Lys-containing peptide complexes in the gas phase was found to be almost as strong as that of Arg-containing complexes. Both systems showed stronger binding than His- containing peptides studied previously. To probe the structure of Arg and Lys non-covalent complexes (charge solvation versus salt bridges), two techniques, CID and ionmolecule reactions, were used. CID experiments indicate that the gas-phase complexes are most likely formed by charge solvation of the central metal ion in the metalloporphyrin by basic side chains of Arg or Lys. Results from the ionmolecule reaction studies are consistent with the charge solvation structure as well.     

Simultaneous quantitation of 17 female sex hormones in illegal products by validated liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry methods

The consumption of food and drugs adulterated with female sex hormones can have an extremely adverse effect on human health. Therefore, developing appropriate monitoring methods for the identification of various exogenous female sex hormones is crucial for minimizing and eliminating the related health risks. Herein, 17 female hormones categorized into two groups: estrogen and progestin, were analyzed using reversed-phase liquid chromatography coupled to Orbitrap or triple quadrupole mass spectrometry. The fragmentation patterns for all compounds were discovered, and fragmented structures were also derived from them through liquid chromatography–high-resolution mass spectrometry followed by qualitative sample analysis. In addition, a quantitative analysis of 67 samples of illicit drugs and dietary supplements was performed using the validated liquid chromatography-tandem mass spectrometry method. Female hormone components were detected in two samples of an unauthorized injectable solution and a tablet-type drug. Medroxyprogesterone was detected in the samples in the range of 96.4–206 ng/g. Notably, eight components similar in structure to steroids were simultaneously detected as male sex hormones by confirming their fragmentation ion patterns using liquid chromatography–high-resolution mass spectrometry. The developed methods thus offer a dependable and practically applicable approach for the screening and detection of exogenous female sex hormones in real food and drug samples to ensure public health.     

Probing RNA hairpins with cobalt(III)hexammine and electrospray ionization mass spectrometry

In this work, electrospray ionization mass spectrometry (ESI MS) was employed to study the interactions of cobalt(III) hexammine, Co(NH3)6(3+), with five RNA hairpins representing the 790 loop of 16S ribosomal RNA and 1920 loop of 23S ribosomal RNA. The RNAs varied in mismatch identity (G.U versus A.C) and level of base modification (pseudouridine versus uridine). Co(NH3)6(3+) binding was observed with the four RNA hairpins that contained a G.U wobble pair in the stem region. ESI MS revealed 1:1 and 1:2 complex formation with all RNAs. Weaker binding was observed with the fifth RNA hairpin that contained an A.C wobble pair in the stem region. The effects of pH on Co(NH3)6(3+) binding were also examined.     

Monitoring of betulin nanoemulsion treatment and molecular changes in mouse skin cancer using surface enhanced Raman spectroscopy

The aim of this study was to obtain in vivo and ex vivo reproducible surface enhanced Raman signal from mouse skin and to use it for the differentiation of skin pathologies. We induced skin carcinoma in mice models using a chemical treatment and tested the chemopreventive activity of a new formulation based on natural compound betulin extracted from the bark of birch trees. Using surface enhanced Raman spectroscopy (SERS) we identified in vivo and ex vivo the spectral signatures characteristic to the healthy skin, melanoma skin induced in mouse models, and to the pathology evolution when the betulin nanoemulsion formulation was topically applied on the cancerous skin of mice. SERS markers associated to each pathology were identified and the signal was distinguished from the classical Raman signal of skin based on several biomarkers, such as the disappearance of the amide I band of proteins, the amplification of the 1574 cm⁻¹ band assigned to nucleic acid bases, and the appearance of the highly amplified band at 230 cm⁻¹ characteristic to the metal-biomolecules complex. The various skin pathologies were differentiated using principal components analysis and K-means clustering. The effectiveness of the betulin nanoemulsion treatment was validated by the histological examination and the chemometrics methods, which successfully confirmed the direct SERS differentiation between the cancerous and the betulin nanoemulsion treated skin.     

Probing the kinetics in supramolecular chemistry and molecular assembly by microfluidic-NMR spectroscopy

Microfluidic-NMR spectroscopy has been extended to study the kinetics in supramolecular chemistry and molecular assembly. Kinetics of a multicomponent host-guest supramolecular system containing viologen derivatives, β-cyclodextrins and cucurbit [7]urils are studied by a PMMA based microfluidic chip combined with a dedicated transmission line probe for NMR detection. By combining microfluidic technology with NMR spectroscopy, the amount of material required for a full kinetic study could be minimized. This is crucial in supramolecular chemistry, which often involves highly sophisticated and synthetically costly building blocks. The small size of the microfluidic structure is crucial in bringing the time scale for kinetic monitoring down to seconds. At the same time, the transmission line NMR probe provides sufficient sensitivity to work at low (2 mM) concentrations.     

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-β-D-glucoside, emodin-8-O-β-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.