At the beginning of 2015 our forensic group published a critical review in Analytical Chemistry titled "Vibrational Spectroscopy: Recent Developments to Revolutionize Forensic Science".
Forensic science is intimately involved in judicial systems, and as such it must be completely objective and reliable. Because forensics is so diverse and extensive, it can be difficult to hold the entire field to this standard. The National Academy of Sciences published a report outlining the current state of forensic science in the U.S., including issues being faced and necessary changes. Raman and infrared (IR) spectroscopy are becoming increasingly more popular in forensic science. Both methods are nondestructive, rapid, quantitative, and confirmatory. Raman spectroscopy, in particular, is known for its intrinsically selective nature.2 It has also been suggested that it is “suited to be the process control star of the next century.”3 These qualities, along with their automated capabilities, make Raman and IR spectroscopy model techniques according to the requirements outlined by the National Academy of Sciences.
Raman hyperspectroscopy combined with advanced statistics is uniquely suitable for characterizing microheterogeneous systems. Understanding the structure and biochemical composition of samples at the microscopic level is important for many practical applications, including various bioanalytical tasks such as medical diagnostics.
The great potential of Raman hyperspectroscopy for medical diagnostics is based on its ability to integrate the impact of multiple biomarkers in a specific spectroscopic signature. Multiple spectra are collected from individual points of a sample, which represent approximately a few hundred femtoliters of volume. The collected spectral data contributes to a three-dimensional hyperspectral data cube (x, y, λ), where x and y are spatial dimensions and λ is the spectral dimension. When machine learning is applied to the data cube, the spatial distribution of biochemical components can be elucidated. This produces a statistically significant characterization of the sample’s heterogeneity and multicomponent composition which can further be used to identify biomarkers, including those present at low average concentrations. Since the entirety of the biochemical composition of a sample is probed, the spectroscopic signature produced for different disease states will be based on the simultaneous integration of multiple biomarkers of the disease, which significantly improves the sensitivity and selectivity of the technique for diagnostic purposes. Thus the combination of Raman hyperspectroscopy and machine learning has powerful analytical applications, including medical diagnostics.
The Lednev lab has used this approach for successfully identifying Alzheimer’s disease in various body fluids including blood serum (link: https://doi.org/10.1002/jbio.201400060 and https://doi.org/10.3390/app9163256) and (link: https://content.iospress.com/articles/journal-of-alzheimers-disease/jad190675). Current efforts are focused on understanding the ability of the methodology to diagnose other diseases including Duchenne Muscular Dystrophy, Diabetes, Celiac Disease, and Lyme disease. The method has the potential to be applied toward identifying any disease which exhibits pathophysiological changes.
Amyloid fibrils are large aggregates of misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and vascular dementia. The amount of hydrogen sulfide (H2S) is known to be significantly reduced in the brain tissue of people diagnosed with Alzheimer’s disease relative to that of healthy individuals. These findings prompted us to investigate the effects of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL). HEWL forms typical β-sheet rich fibrils during the course of 70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of β-sheet and amyloid fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy and ThT fluorescence.
We recently discovered that hydrogen sulfide disrupts the formation of fibrils formed from lysozyme. Rosario-Alomar et al. J Phys Chem B
Nonresonance Raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550–500 cm–1 spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm–1 band assigned to the trisulfide group (RSSSR) based on the comparison with model compounds. The formation of RSSSR was proven further using a reaction with TCEP reduction agent and LC-MS analysis of the products. Intrinsic tryptophan fluorescence study shows a strong denaturation of HEWL containing trisulfide bonds. The presented evidence indicates that H2S causes the formation of trisulfide bridges, which destabilizes HEWL structure, preventing protein fibrillation. As a result, small spherical aggregates of unordered protein form, which exhibit no cytotoxicity by contrast with HEWL fibrils.
The long-term goal of our research is to advance the deep UV resonance Raman (DUVRR) spectroscopic methodology to the level of a reliable and simple analytical and bioanalytical tool for quantitative structural characterization of small molecules and biological systems. This includes the development of (i) novel instrumentation and (ii) the most advanced statistical analysis based on chemometrics and 2D correlation spectroscopy and molecular modeling. Our current research projects include the investigation of amyloid fibril formation, structural rearrangements of proteins involved into intracellular signaling process, and application of deep UV Raman spectroscopy for environmental and forensic purposes.
The irradiation of a molecular system with a monochromatic light always results in two types of light scattering, elastic and inelastic scattering. Elastic scattering, which occurs with no change in photon frequency, is called Rayleigh scattering. Raman scattering is accompanied by the shift in photon frequency due to excitation/deactivation of molecular vibrations. Raman spectrum provides a vibrational signature of the molecular system and, consequently, the information about molecular structure. Normal Raman scattering is relatively inefficient process. The efficiency and selectivity of Raman scattering could be dramatically improved due to the resonance enhancement, which occurs when the excitation wavelength is within an electronic transition of the molecular system. Further increase in Raman spectroscopic efficiency occurs when ultraviolet(UV) excitation is used.
Raman spectroscopy provides a NONINVASIVE way to characterize STRUCTURE and DYNAMICS of biological systems under PHYSIOLOGICAL CONDITIONS.
Time-resolved Raman spectroscopy is an effective tool for real-time kinetic studies of protein folding. We have built the first nanosecond time-resolved UV Raman spectrometer combined with a laser induced temperature jump technique. (Lednev et al. JACS 1999, 4076 and 8074, JACS 2001, 2388) We plan to extend further this methodology to initiate protein folding with a pH jump and an ion-concentration jump to study proteins involved in cellular signaling processes.
Raman spectroscopy provides unique opportunities for development of remote sensing systems for environmental purposes including nuclear waste testing.
This is based upon:
- the striking difference of Raman signatures for different chelating agents
- substantial changes in the chelating agent Raman spectra in the analyte presence
- an enormous increase in sensitivity due to a surface enhanced Raman effect
- modern high throughput fiber optics, inexpensive lasers, and CCD detectors.
A new deep UV Raman spectroscopic apparatus (Lednev et al. Anal Bioanal Chem 2005, 431) has been recently designed, built and characterized at Albany. As a light source, the apparatus utilizes either (i) Indigo S laser system from Coherent allowing for tuning the excitation between 193 and 205 nm, or (ii) Powerlight 9050 laser system from Continuum combined with homebuilt Raman shifter. The apparatus requires only a 100-µl sample with protein concentration as low as 0.1 mg/ml. No special sample preparation is required: the dynamic range has no limitations at the high concentration end, although self-absorption might need to be taken into account for quantitative analysis of Raman spectra. Extending the excitation wavelength deeper into the UV region allows for resonantly enhancing Raman scattering from an amide chromophore, a building block of a polypeptide backbone, that does not exhibit electronic absorption in the near UV and visible spectral range. Amide chromophore Raman spectra provide quantitative information about the secondary structure of proteins. The high sensitivity of deep UV resonance Raman spectroscopy makes this nondestructive method of analysis especially valuable for studying biological systems under physiological conditions. Near UV Raman spectroscopy with excitation at and above 228 nm has already found applications in biology for characterizing protein structure and dynamics.
Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the conformational evolution, i.e., the transformation of native protein to the highly ordered cross-ß structure is still under active investigation. Protein structural transformations on the molecular level during in vitro fibril formation are accompanied by substantial changes in macroscopic properties, such as formation of a gelatinous phase and the formation of insoluble particles. These changes limit the application of conventional methods such as NMR, SAXS, CD, FTIR, intrinsic and ANS fluorescence, etc. for characterizing protein conformational transformations. Raman spectroscopy has been proven to be an efficient technique for characterizing highly-scattering and opaque samples.
We utilize DUVRR spectroscopy, along with other spectroscopic techniques, including tryptophan fluorescence, circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) to study quantitatively the structural evolution during in vitro fibrillation of hen egg white lysozyme (Xu et al. Biopolymers 2005, 58). Lysozyme formed fibril under prolonged incubation in acidic solution at 65ºC. Unlike fully reversible denaturation of lysozyme caused by brief heating, which resulted in only ~15% of a-helix melting at 65ºC, the prolonged incubation at this temperature caused much larger and irreversible structural changes of the protein. The resulting partially denatured intermediate contained ~8% of a-helix and ~8% of ß-sheet as compared with 32% and 6% of a-helix and ß-sheet, respectively, in the native protein. Both secondary and tertiary structures evolved in a monoexponential fashion with characteristic time of about 30 hours. DUVRR spectroscopy was shown to be superior over the far-UV CD spectroscopy in the quantitative study of fibril formation: DUVRR spectroscopy (i) was capable of characterizing inhomogeneous, highly light scattering samples containing fibrils and (ii) was highly sensitive to the formation of ß-sheet conformation. In addition, phenylalanine was shown to be an informative DUVRR spectroscopic biomarker of protein structural rearrangements during fibril formation.
(A) A three-component composition of the solution part of lysozyme incubated at 65 ºC and pH 2. (B) A relative intensity of the 1000-cm-1phenylalanine band in the Raman spectra of lysozyme solutions incubated for various times. I0 corresponds to the band intensity in the spectrum of a non-incubated solution. I value was normalized to the protein concentration in solutions. (C) The percentage of lysozyme deposited in a gelatinous form. The solid curves represent monoexponential fits.
A de novo 687-amino acid residue polypeptide (Topilina et al. Biomacromolecules 2006, 1104) with a regular 32 amino acid repeat sequence, (GA)3GY(GA)3GE(GA)3GH(GA)3GK, forms large ß-sheet assemblages which exhibit remarkable folding properties and, as well, form fibrillar structures (Lednev et al. Biophys. J. 2006, 3805). This construct is an excellent tool to explore the details of ß-sheet formation yielding intimate folding information which is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel ß-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 °C adopting a predominant PPII conformation. Deep UV Raman spectroscopy indicated melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of ~1 and ~60 min, respectively, indicating no detectable formation of hairpin-type nuclei in millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of ß-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less-frustrated free energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.
2.5 repeats of (GA)3GY(GA)3GE(GA)3GH(GA)3GK constituent polypeptide unit of YEHK21
Kinetics of YEHK21 melting and folding initiated with temperature change. (a) DUVRR spectra of YEHK21 measured 20, 60 and 340 sec after the 25 to 85 °C temperature jump. The spectra were measured in an open solution stream. The accumulation time was 20 sec. The difference spectra (b) and (c) were obtained by subtracting DUVRR spectra of YEHK21 measured 20 and 340 sec after the temperature jump from that measured before the jump. (d) The difference between the room-temperature DUVRR spectra of YEHK21 obtained for the initial folded polypeptide and that briefly heated to 125 °C and cooled to the room temperature (see Fig. 5). (g) DUVRR spectra of YEHK21 measured 1, 60 and 240 min at the room temperature after a 5-min exposure to 100 °C. The spectra were measured in an NMR tube. The accumulation time was 60 sec. The spectrum of the initial folded polypeptide (black) is shown for comparison. The difference spectra (e) and (f) were obtained by subtracting the DUVRR spectrum of the initial folded YEHK21 from those measured 60 and 240 min at the room temperature after a brief exposure to 100 °C.
DUVRR spectroscopy combined with chemometric analysis was shown to be a powerful tool for quantitative characterization of multiple equilibria between lutetium and a bicyclic diamide (Shashilov et al. Inorg Chem 2006, 3606). Several chemometric methods were utilized for a comparative analysis of Raman spectroscopic data. It was found that a recently developed stepwise maximum angle calculation (SMAC) algorithm followed by alternative least squares (ALS) was more efficient than the commonly used combination of evolving factor analysis (EFA) and ALS methods, especially when little or no information about the system composition and the spectra of individual components was available. Complex formation between a bicyclic diamide, a novel chelating agent for lanthanides and actinides, and lutetium in acetonitrile solution was investigated. A free ligand and its lutetium complexes showed weak, non-characteristic near-UV absorption and no fluorescence that limited the application of absorption and fluorescence spectroscopies for studying this system. A free ligand and 1:1, 1:2 and 1:3 metal:ligand complexes were distinguished in a bicyclic diamide/lutetium solution. The composition evolution of the solution during the course of titration with lutetium was described, and the stepwise stability constants of complex formation, (K1:K2)=0.80±0.15, (K1,2>10ˆ8 Mˆ-1) and K3=(5.5±1)•10ˆ3 Mˆ-1, were estimated.
Latent variable analysis of DUVRR spectra was demonstrated to be a powerful tool for characterizing protein secondary structural composition (Shashilov et al. JQSRT 2006, 46). Non-negative independent component analysis (ICA) and pure variable methods, such as stepwise maximum angle calculation (SMAC) and simple-to-use interactive self-modeling mixture analysis (SIMPLISMA), were employed for examination of ten DUVRR spectra of lysozyme obtained at various stages of its partial denaturation, the first stage of amyloid fibril formation. The non-negative ICA allowed for extracting the spectrum of the ß-sheet from deep UV resonance Raman spectra of lysozyme while principle component analysis (PCA) and multivariate curve resolution (MCR) could not separate the ß-sheet constituent as an individual component. No initial guess about the features of the ß-sheet spectrum was used. Pure variable methods SMAC and SIMPLISMA were found to resolve three independent spectral components assigned to ß-sheet, random coil, and native lysozyme.
Molecular modeling, which included structure optimization and calculation of Raman frequencies and resonance intensities, allowed for assigning all strong Raman bands of the bicyclic diamide as well as predicting the band shifts observed due to complex formation with metal ions. A comparative analysis of Raman spectra and the results of the molecular modeling could be used for elucidating the structure of complexes in solution. DUVRR spectroscopy was used for characterizing ligand-metal ion complexes. The obtained results demonstrated a strong intrinsic sensitivity and selectivity of a Raman spectroscopic signature of a bicyclic diamide, a novel chelating agent for lanthanides and actinides recently reported by James Hutchison with coworkers (JACS 2002). We are very grateful to Prof. James E. Hutchison and Ms. Bevin W. Parks from the University of Oregon for providing bicyclic diamides for this study.