Paul M. Donaldson

Biological and Biomedical Applications of Two-Dimensional Vibrational Spectroscopy: Proteomics, Imaging, and Structural Analysis

In the last 10 years, several forms of two-dimensional infrared (2DIR) spectroscopy have been developed, such as IR pump-probe spectroscopy and photon-echo techniques. In this Account, we describe a doubly vibrationally enhanced four-wave mixing method, in which a third-order nonlinear signal is generated from the interaction of two independently tunable IR beams and an electron-polarizing visible beam at 790 nm. When the IR beams are independently in resonance with coupled vibrational transitions, the signal is enhanced and cross-peaks appear in the spectrum. This method is known as either DOVE (doubly vibrationally enhanced) four-wave mixing or EVV (electron-vibration-vibration) 2DIR spectroscopy. We begin by discussing the basis and properties of EVV 2DIR. We then discuss several biological and potential biomedical applications. These include protein identification and quantification, as well as the potential of this label-free spectroscopy for protein and peptide structural analysis. In proteomics, we also show how post-translational modifications in peptides (tyrosine phosphorylation) can be detected by EVV 2DIR spectroscopy. The feasibility of EVV 2DIR spectroscopy for tissue imaging is also evaluated. Preliminary results were obtained on a mouse kidney histological section that was stained with hematoxylin (a small organic molecule). We obtained images by setting the IR frequencies to a specific cross-peak (the strongest for hematoxylin was obtained from its analysis in isolation; a general CH(3) cross-peak for proteins was also used) and then spatially mapping as a function of the beam position relative to the sample. Protein and hematoxylin distribution in the tissue were measured and show differential contrast, which can be entirely explained by the different tissue structures and their functions. The possibility of triply resonant EVV 2DIR spectroscopy was investigated on the retinal chromophore at the centre of the photosynthetic protein bacteriorhodopsin (bR). By putting the visible third beam in resonance with an electronic transition, we were able to enhance the signal and increase the sensitivity of the method by several orders of magnitude. This increase in sensitivity is of great importance for biological applications, in which the number of proteins, metabolites, or drug molecules to be detected is low (typically pico- to femtomoles). Finally, we present theoretical investigations for using EVV 2DIR spectroscopy as a structural analysis tool for inter- and intramolecular interaction geometries.

Enhancing signal detection and completely eliminating scattering using quasi-phase-cycling in 2D IR experiments

We demonstrate how quasi-phase-cycling achieved by sub-cycle delay modulation can be used to replace optical chopping in a box-CARS 2D IR experiment in order to enhance the signal size, and, at the same time, completely eliminate any scattering contamination. Two optical devices are described that can be used for this purpose, a wobbling Brewster window and a photoelastic modulator. They are simple to construct, easy to incorporate into any existing 2D IR setup, and have attractive features such as a high optical throughput and a fast modulation frequency needed to phase cycle on a shot-to-shot basis.

2D-IR study of a photoswitchable isotope-labeled alpha-helix.

A series of photoswitchable, alpha-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with (13)C(18)O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single (13)C(18)O label can give rise to two bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other from a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and unfolded state of the helix. For most sites, unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives an indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we conclude that the helix is not uniformly folded. Furthermore, the shift in hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.

Protein identification and quantification by two-dimensional infrared spectroscopy: implications for an all-optical proteomic platform.

Electron-vibration-vibration two-dimensional coherent spectroscopy, a variant of 2DIR, is shown to be a useful tool to differentiate a set of 10 proteins based on their amino acid content. Two-dimensional vibrational signatures of amino acid side chains are identified and the corresponding signal strengths used to quantify their levels by using a methyl vibrational feature as an internal reference. With the current apparatus, effective differentiation can be achieved in four to five minutes per protein, and our results suggest that this can be reduced to <1 min per protein by using the same technology. Finally, we show that absolute quantification of protein levels is relatively straightforward to achieve and discuss the potential of an all-optical high-throughput proteomic platform based on two-dimensional infrared spectroscopic measurements.

Gold Nanoparticle Capping Layers: Structure, Dynamics, and Surface Enhancement Measured Using 2D-IR Spectroscopy†

Surface analysis: two-dimensional infrared spectroscopy (2D-IR) was used to probe amide and carboxylate functional groups in the vicinity of gold nanoparticles and their aggregates. The data presented shows that the unique ability of 2D-IR spectroscopy to characterize broadening mechanisms and cross peaks offers new insights into gold nanoparticle capping layer structure and dynamics.

Direct identification and decongestion of Fermi resonances by control of pulse time ordering in two-dimensional IR spectroscopy

We show that it is possible to both directly measure and directly calculate Fermi resonance couplings in benzene. The measurement method used was a particular form of two-dimensional infrared spectroscopy (2D-IR) known as doubly vibrationally enhanced four wave mixing. By using different pulse orderings, vibrational cross peaks could be measured either purely at the frequencies of the base vibrational states or split by the coupling energy. This capability is a feature currently unique to this particular form of 2D-IR and can be helpful in the decongestion of complex spectra. Five cross peaks of the ring breathing mode nu13 with a range of combination bands were observed spanning a region of 1500-4550 cm(-1). The coupling energy was measured for two dominant states of the nu13+nu16 Fermi resonance tetrad. Dephasing rates were measured in the time domain for nu13 and the two (nu13+nu16) Fermi resonance states. The electronic and mechanical vibrational anharmonic coefficients were calculated to second and third orders, respectively, giving information on relative intensities of the cross peaks and enabling the Fermi resonance states of the combination band nu13+nu16 at 3050-3100 cm(-1) to be calculated. The excellent agreement between calculated and measured spectral intensities and line shapes suggests that assignment of spectral features from ab initio calculations is both viable and practicable for this form of spectroscopy.

Vibrational energy transport through a capping layer of appropriately designed peptide helices over gold nanoparticles.

We design and characterize spherical gold nanoparticles, which are covalently linked to and completely covered by 3(10)-helical peptides. These helices provide a scaffold to place (13)C=O isotope labels at defined distances from the gold surface, which we employ as local thermometers. Probing these reporter groups with transient infrared spectroscopy, we monitor the vibrational energy flow across the peptide capping layer following excitation of the nanoparticle plasmon resonance.

Broadband near-field infrared spectromicroscopy using photothermal probes and synchrotron radiation

In this paper, we experimentally demonstrate the use of infrared synchrotron radiation (IR-SR) as a broadband source for photothermal near-field infrared spectroscopy. We assess two methods of signal transduction; cantilever resonant thermal expansion and scanning thermal microscopy. By means of rapid mechanical chopping (50-150 kHz), we modulate the IR-SR at rates matching the contact resonance frequencies of atomic force microscope (AFM) cantilevers, allowing us to record interferograms yielding Fourier transform infrared (FT-IR) photothermal absorption spectra of polystyrene and cyanoacrylate films. Complementary offline measurements using a mechanically chopped CW IR laser confirmed that the resonant thermal expansion IR-SR measurements were below the diffraction limit, with a spatial resolution better than 500 nm achieved at a wavelength of 6 μm, i.e. λ/12 for the samples studied. Despite achieving the highest signal to noise so far for a scanning thermal microscopy measurement under conditions approaching near-field (dictated by thermal diffusion), the IR-SR resonant photothermal expansion FT-IR spectra measured were significantly higher in signal to noise in comparison with the scanning thermal data.

A 100 kHz Time-Resolved Multiple-Probe Femtosecond to Second Infrared Absorption Spectrometer

We present a dual-amplifier laser system for time-resolved multiple-probe infrared (IR) spectroscopy based on the ytterbium potassium gadolinium tungstate (Yb:KGW) laser medium. Comparisons are made between the ytterbium-based technology and titanium sapphire laser systems for time-resolved IR spectroscopy measurements. The 100 kHz probing system provides new capability in time-resolved multiple-probe experiments, as more information is obtained from samples in a single experiment through multiple-probing. This method uses the high repetition-rate probe pulses to repeatedly measure spectra at 10 µs intervals following excitation allowing extended timescales to be measured routinely along with ultrafast data. Results are presented showing the measurement of molecular dynamics over >10 orders of magnitude in timescale, out to 20 ms, with an experimental time response of <200 fs. The power of multiple-probing is explored through principal component analysis of repeating probe measurements as a novel method for removing noise and measurement artifacts.

Detection of complex formation and determination of intermolecular geometry through electrical anharmonic coupling of molecular vibrations using electron-vibration-vibration two-dimensional infrared spectroscopy

Electrical interactions between molecular vibrations can be non-linear and thereby produce intermolecular coupling even in the absence of a chemical bond. We use this fact to detect the formation of an intermolecular complex using electron-vibration-vibration two-dimensional infrared spectroscopy (EVV 2DIR) and also to determine the distance and angle between the two molecular species.