Non-resonance micro-Raman spectroscopic studies on crystalline domoic acid and its aqueous solutions

Domoic acid (DA) is a neurotoxin naturally present in the marine ecosystem. Since DA's toxicity has been explained by its molecular structure and particularly because of its ethylenic double bond, spectroscopic investigation of this molecule is of importance. We carried out Raman spectroscopy on crystalline DA and on DA in aqueous solutions (28,000-25 ng DA/mL) and assigned Raman modes in comparison with the Raman spectra of its substructures. Noise-free, clear Raman signal from the solutions containing low concentrations of DA were obtained by applying the drop coating deposition Raman (DCDR) technique. Raman spectra reveal that crystalline DA exists in the zwitterionic form. The Raman spectra of the DA aqueous solutions were analysed in the light of their pH whereas the variation in the spectra was attributed to the hydration, the degree of protonation and crystallinity of the solid film. We show that DCDR can be applied for the rapid detection of domoic acid down to 25 ng DA/mL (0.025 ppm).     

Analysis of human tear fluid by Raman spectroscopy

Tear fluid is a complex aqueous solution containing proteins, metabolites, electrolytes and lipids. This study uses Raman spectroscopy to analyse the composition of human tear fluid from three healthy volunteers. Two different methods are used to obtain Raman spectra from the 3 μL tear samples: (i) solution-phase Raman spectroscopy, and (ii) drop coating deposition Raman spectroscopy (DCDRS). Tear samples were either basal fluid, or yawn reflex secreted fluid. Calibration of the solution technique with standard protein solutions (5-15 mg mL⁻¹) showed that this method could predict the protein concentration (cross-validation) with an error of less than 1 mg mL⁻¹. The Raman signals from the tear fluid were very weak but signals due to protein and urea were clearly observable in all samples. The drop coating deposition technique was shown to produce very high signal-to-noise spectra for relatively short acquisition times, and small sample volumes. Raman point mapping combined with principal components analysis showed that the protein, urea, bicarbonate and lipid could all be detected in the tear samples and that the distribution of these components was inhomogeneous. Their position within the drying pattern was shown to depend on their relative solubilities. The results of this study suggest that solution Raman measurements may be calibrated to give the total tear protein concentration and DCDRS could be used to give a fingerprint of the tear protein (and lipid) composition.     

Detection of the site of phosphorylation in a peptide using Raman spectroscopy and partial least squares discriminant analysis

Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13-residue peptide whose sequence derives from the cellular protein pp60(c-src) (protein tyrosine kinase). Raman spectra of serine, threonine and tyrosine amino acids and their phosphorylated derivatives are used to aid in the interpretation of peptide spectra. The purity of the synthetic peptides are confirmed by mass spectroscopy. Peptide Raman measurements are performed using the recently reported drop-coating deposition Raman (DCDR) method, followed by Savistky-Golay second derivative (SGSD) pre-processing and multivariate spectral classification using partial least squares (PLS) discriminant analysis. Leave-one-out training/testing results are displayed using a PLS psuedo-probability score plot and shown to facilitate error-free spectral determination of the site of phosphorylation.     

Quartz crystal microbalance study of protein adsorption kinetics on poly(2-hydroxyethyl methacrylate)

The interaction of macromolecules with artificial biomaterials may lead to potentially serious complications upon implantation into a biological environment. The interaction of one of the most widely used biomaterials, polyHEMA, with lysozyme, bovine serum albumin (BSA), and lactoferrin was investigated using quartz crystal microbalance (QCM). The concentration dependence of adsorption was measured for the aforementioned proteins individually as well as for lysozyme-BSA, and lysozyme-lactoferrin combinations. An extension of Voinova's viscoelastic model to n layers was used to create thickness-time graphs for adsorption. For each of lactoferrin and lysozyme, two distinctly different timescales of adsorption could be differentiated. However, the mechanisms of adsorption appeared to differ between the two. Negative dissipation shifts were measured for low concentrations of lysozyme, trending to positive dissipation at higher concentrations. This suggested that lysozyme was adsorbed initially into the matrix, stiffening the hydrogel, and later onto the surface of polyHEMA. Additionally, trials with commercial no-rub cleaning solutions indicated little added effectiveness over buffer solutions. Mixtures of proteins showed behaviour which differed in some cases from the simple combination of single protein adsorption experiments.     

Effect of polysaccharide structure on protein adsorption

Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and γ-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces.     

Molecular packing of lysozyme,fibrinogen, and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy

Infrared-visible sum frequency generation (SFG) vibrational spectroscopy, in combination with fluorescence microscopy, was employed to investigate the surface structure of lysozyme, fibrinogen, and bovine serum albumin (BSA) adsorbed on hydrophilic silica and hydrophobic polystyrene as a function of protein concentration. Fluorescence microscopy shows that the relative amounts of protein adsorbed on hydrophilic and hydrophobic surfaces increase in proportion with the concentration of protein solutions. For a given bulk protein concentration, a larger amount of protein is adsorbed on hydrophobic polystyrene surfaces compared to hydrophilic silica surfaces. While lysozyme molecules adsorbed on silica surfaces yield relatively similar SFG spectra, regardless of the surface concentration, SFG spectra of fibrinogen and BSA adsorbed on silica surfaces exhibit concentration-dependent signal intensities and peak shapes. Quantitative SFG data analysis reveals that methyl groups in lysozyme adsorbed on hydrophilic surfaces show a concentration-independent orientation. However, methyl groups in BSA and fibrinogen become less tilted with respect to the surface normal with increasing protein concentration at the surface. On hydrophobic polystyrene surfaces, all proteins yield similar SFG spectra, which are different from those on hydrophilic surfaces. Although more protein molecules are present on hydrophobic surfaces, lower SFG signal intensity is observed, indicating that methyl groups in adsorbed proteins are more randomly oriented as compared to those on hydrophilic surfaces. SFG data also shows that the orientation and ordering of phenyl rings in the polystyrene surface is affected by protein adsorption, depending on the amount and type of proteins.     

Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry

The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on-line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears.     

Protein films adsorbed on experimental dental materials: ToF-SIMS with multivariate data analysis

The proteins lysozyme, amylase, and bovine serum albumin (BSA) were adsorbed on two experimental dental materials, made of fluoroapatite particles embedded in polymer matrices, and on silicon wafers. The protein films were prepared as single-component layers, as binary mixtures, and as double layers. These systems were investigated by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the multivariate data analysis technique of discriminant principal-component analysis (DPCA). During adsorption of a single protein film on to the solid surfaces, the three proteins could be clearly distinguished by the scores of their mass spectra after selection of amino acid-related peaks and DPCA. Furthermore, very similar results were obtained on the two different fluoroapatite substrates. For samples coated with binary layers of two proteins adsorbed simultaneously, it was found for both substrate types that BSA shows the strongest ability to adsorb followed by lysozyme, while amylase has the smallest ability. By contrast, the consecutive adsorption of two protein layers showed a strong influence of substrate type on the adsorption ability of the proteins.     

In situ Detection of Amide A Bands of Proteins in Water by Raman Ratio Spectrum

The amide A band of protein is sensitive to the hydrogen bands of amide groups of proteins. However, it is hard to distinguish the amide A band of aqueous protein in situ directly, since it overlaps with O-H stretching vibration of water. In this work, we presented a new analytical method of Raman ratio spectrum, which can extract the amide A band of proteins in water. To obtain the Raman ratio spectrum, the Raman spectrum of aqueous protein was divided by that of pure water. A mathematical simulation was employed to examine whether Raman ratio spectrum is effective. Two kinds of protein, lysozyme and α-chymotrypsin were employed. The amide A bands of them in water were extracted from Raman ratio spectra. Additionally, the process of thermal denaturation of lysozyme was detected from Raman ratio spectrum. These results demonstrated the Raman ratio spectra could be employed to study the amide A modes of proteins in water.     

Quantitative histochemical analysis of human artery using Raman spectroscopy.

We have developed a method for using near infrared Raman spectroscopy to quantitatively analyze the histochemical composition of human artery. The main contributors to bands observed in the Raman spectra of normal and atherosclerotic aorta are the proteins collagen and elastin, cholesterol lipids, and calcium hydroxyapatite. The Raman scattering cross-sections of different bands for these components have been determined in order to understand their relative contributions to the Raman spectra of biological tissue. The Raman signal is observed to behave linearly with the concentration of the components, even in a highly scattering medium such as a powder. Using these data, we have developed a linear model that can be used to extract the quantitative contribution of an individual component to the spectrum of a mixture. The model has been applied to several mixtures of known composition of tissue constituents in order to evaluate its precision and accuracy. The calculated fit coefficients from the spectra are in agreement with the measured values within experimental uncertainties. The spectra of different types of atherosclerotic aorta have also been modeled, and we have extracted quantitative information regarding the relative concentration of biological constituents in atherosclerotic aorta.     

Phase-resolved fluorimetric determination of two albumin-bound fluorescein species

A new approach is described for phase-resolved fluorescence spectroscopy, for use in resolving mixtures of two components with very similar fluorescence spectra and life-times. Results are given for application of the technique to solutions containing fluorescein physically bound to albumin and fluorescein isothiocyanate covalently bound to albumin. Because the two fluorescein species have essentially identical fluorescence spectra and a phase-angle difference of only 2°, the conventional phase-resolved method in which measurements are made at the two phase angles at which the fluorescence contribution from one or the other of the components is zero will not resolve the components. Solutions containing 25–50 nM of each component were successfully resolved by making measurements at two other phase angles and solving the pair of simultaneous equations that is generated. Accuracy is best (average relative error, 3%) using detector phase angles corresponding to a 45° shift from the phase angles of the components. Relative standard deviations of ±16% are obtained at these phase angles. Solutions containing 5–50 nM fluorescein and 50–500 nM fluorescein isothiocyanate conjugated to albumin could also be resolved, with an average relative error of 16% and ±2.4 r.s.d. The method could be used for simultaneous determination of a fluorophore in two different microenvironments, as in protein-ligand binding studies and in homogeneous immunoassay.     

Quantification of casein phosphorylation with conformational interpretation using Raman spectroscopy

Raman spectroscopy is emerging as a powerful method for obtaining both quantitative and qualitative information from biological samples. One very interesting area of research, for which the technique has rarely been used, is the detection, quantification and structural analysis of post-translational modifications (PTMs) on proteins. Since Raman spectra can be used to address both of these questions simultaneously, we have developed near infrared Raman spectroscopy with appropriate chemometric approaches (partial least squares regression) to quantify low concentration (4 microM) mixtures of phosphorylated and dephosphorylated bovine alpha(s)-casein. In addition, we have used these data in conjunction with Raman optical activity (ROA) spectra and NMR to assess the structural changes that occur upon phosphorylation.     

The adsorbed conformation of globular proteins at the air/water interface

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and beta-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and beta-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little change to their native secondary structure.     

Determination of human serum albumin and γ-globulin in a control serum solution by near-infrared spectroscopy and partial least squares regression

Near infrared (NIR) spectra in the 1300– 1850 nm region were measured for control serum solutions containing both albumin and γ-globulin of various concentrations. Partial least squares two (PLS2) regression was applied to the NIR spectra to determine simultaneously the concentrations of both proteins. For albumin, the correlation coefficient (R) of 0.988, the standard error of calibration (SEC) of 1.61 g/L, the standard error of prediction (SEP) of 1.29 g/L, the relative standard deviation (RSD) of 0.026 and the ratio of standard deviation of reference data in prediction to SEP (RPD) of 12.2 were obtained. For γ-globulin, the corresponding values were 0.997, 1.36 g/L, 1.35 g/L, 0.0365 and 8.66, respectively. The regression coefficients (RCs) of PLS factors were compared between albumin and γ-globulin, and the observed differences in the RCs were discussed based upon the differences in the hydration between albumin and γ-globulin. In order to explore the effects of various metabolites such as glucose, and cholesterol on the chemometrics models, the RCs for albumin and γ-globulin in the control serum solutions were also compared with those for albumin and γ-globulin in phosphate buffer solutions previously studied. The results of our experiments show that NIR spectroscopy with the use of PLS2 regression has considerable promise in nondestructive determination of the concentrations of blood serum proteins.     

Calorimetric and structural investigation of the interaction of lysozyme and bovine serum albumin with poly(ethylene oxide) and its copolymers

This work reports investigations aiming at verifying the occurrence of specific interactions between lysozyme or bovine serum albumin (BSA) and poly(ethylene oxide) and its copolymers with poly(propylene oxide). Thermal stability of these proteins, followed by means of high sensitivity DSC, was found to be mostly unaffected by the presence of these polymers. Chromatographic experiments (reverse-phase HPLC and size exclusion chromatrography) did not reveal any sign of specific interaction for these mixtures, either. Isothermal titration calorimetry revealed an increase in enthalpy for the mixtures, represented by a positive enthalpy of transfer for these proteins from buffer to polymer solutions. Moreover, SAXS analyses confirmed that at ambient temperatures these polymers do not affect lysozyme structure. In summary, no evidence is found to support earlier suggestions that some kind of complex could be formed between these proteins and poly(ethylene oxide) or its copolymers, but the present results suggest the occurrence of entropically driven hydrophobic effects.     

Cascading third-order Raman process and local structure formation in binary liquid mixtures of benzene and n-hexane

The cascading third-order Raman process in binary mixtures of benzene and n-hexane was studied by six-wave mixing coherent anti-Stokes Raman scattering spectroscopy. By examining the concentration dependence of the cascading third-order signal intensity, we investigated the formation of local structures of benzene in the binary mixtures. A significant deviation from the dependence expected for homogeneous mixtures was observed at benzene concentrations above 7 mol dm(-3). This deviation can be interpreted in terms of optical inhomogeneity caused by the formation of domain structures of benzene molecules. We discuss the feasibility of the cascading third-order process as a sensitive probe for the microscopic structures that are formed in liquids and solutions.     

Characterization of surfactant complex mixtures using Raman spectroscopy and signal extraction methods: Application to laundry detergent deformulation

This paper presents the analysis of surfactants in complex mixtures using Raman spectroscopy combined with signal extraction (SE) methods. Surfactants are the most important component in laundry detergents. Both their identification and quantification are required for quality control and regulation purposes. Several synthetic mixtures of four surfactants contained in an Ecolabel laundry detergent were prepared and analyzed by Raman spectroscopy. SE methods, Independent Component Analysis and Multivariate Curve Resolution, were then applied to spectral data for surfactant identification and quantification. The influence of several pre-processing treatments (normalization, baseline correction, scatter correction and smoothing) on SE performances were evaluated by experimental design. By using optimal pre-processing strategy, SE methods allowed satisfactorily both identifying and quantifying the four surfactants. When applied to the pre-processed Raman spectrum of the Ecolabel laundry detergent sample, SE models remained robust enough to predict the surfactant concentrations with sufficient precision for deformulation purpose. Comparatively, a supervised modeling technique (PLS regression) was very efficient to quantify the four surfactants in synthetic mixtures but appeared less effective than SE methods when applied to the Raman spectrum of the detergent sample. PLS seemed too sensitive to the other components contained in the laundry detergent while SE methods were more robust. The results obtained demonstrated the interest of SE methods in the context of deformulation.     

Zeta potential measurement for air bubbles in protein solutions

Protein adsorption at gas-liquid interfaces is important in a number of processes including foam formation in bioreactors, foam fractionation for protein recovery, and production of protein based food and drinks. The physical properties of the gas-liquid interface will influence foam stability; important properties will include both surface rheological and electrokinetic properties. While surface rheological properties of gas-protein solution interfaces have been reported, there are no published values for electrokinetic properties at such interfaces. In this paper, zeta potential values of gas bubbles in solutions of three proteins, measured using a microelectrophoresis technique, are reported. The three proteins chosen were BSA, beta-casein, and lysozyme; these proteins have all been used previously in protein foaming studies. The effect of protein concentration and ionic strength is considered. For BSA and beta-casein, zeta potential was found to increase with increasing protein concentration and ionic strength. For air bubbles in lysozyme solutions, measured zeta potential was zero. zeta potential values for air bubbles in some binary protein mixtures are also presented.     

Enrichment of proteinaceous materials on a strong cation-exchange diol silica restricted access material: protein-protein displacement and interaction effects

A study of size exclusion and enrichment of proteins employing strong cation-exchange diol silica restricted access material (SCX-RAM) under saturation conditions is presented. Experiments were carried out with bacitracin, protamine, ribonuclease, lysozyme and bovine serum albumin as individual proteinaceous analytes as well as comprehensive binary mixtures and with human urine samples. Protein size dependent capacity features of the SCX-RAM column was observed. Bacitracin demonstrated the highest capacity followed by protamine while adsorption capacities of both ribonuclease and lysozyme were found smaller by a factor of 10. Applying binary protein samples occurring displacement effects were apparent: proteins with strong cationic properties displaced those already adsorbed by the bonded cation-exchange ligands. Bacitracin was displaced in all binary mixture experiments in particular by protamine. Furthermore, the binary mixtures displayed increased adsorption for some proteins due to complex formation. Lysozyme and ribonuclease showed double capacity values when paired with bacitracin. Both phenomena, displacement and enhanced adsorption occurred in the saturated state and led to changes in the urine composition during sample preparation. Injecting urine samples the relative proportions of fractions changed from 4 up to more than 20 times, due to the differences of the protein adsorption capacities on the SCX-RAM column. Analysing urine samples the SCX-RAM column provided extensive long-term stability.