nucleation step of lysozyme fibrillation. ⁵

Significant advances in laser technology, microfluidics, and the development of novel light detectors have dramatically improved spectroscopic methods for molecular characterization over the last decade. In many cases, nanoliter samples are sufficient for high-quality spectroscopic characterization that opens the possibility of working with precious biological and chemical systems. Although the accumulation time is significantly reduced in a typical measurement, the amount of information hidden in digital data sets composed of hundreds and thousands of points is dramatically increased. The once golden rule of previous generations of spectroscopists—that if you do not see a change in the spectrum by naked eye, then you are chasing a ghost—no longer applies. Advanced statistical methods allow the retrieval of qualitative and quantitative information from data sets that is not otherwise evident. For example, we extracted Raman spectroscopic signatures

of protein intermediate states, which form at the early stages of lysozyme fibrillation, by utilizing latent variable analysis without any prior information. ⁶ We also applied DUVRR spectroscopy combined with advanced statistical analysis including two-dimensional (2-D)-correlation spectroscopy, independent component analysis (ICA), and pure variable methods, to study nucleus formation during the fibrillation of hen egg-white lysozyme, a well-studied model of amyloidogenic proteins. ⁷

2-D analysis region

144 h

24 h

Protein characterization using DUVRR Deep-UV excitation (below 200 nm) resonantly enhances Raman scattering from the amide chromophore, a building block of a polypeptide backbone, exhibiting a strong UV absorption peak at approximately 195 nm. The resonance enhancement not only decreases the required sample amount, but also allows for probing specific structural motifs of a protein mole-

Tyr Phe

15 min

Am III

Am II

Am I

Phe

CαH

Spectroscopic Solutions

From Components to Systems

One Complete Source.

Components

1000 1200 1400 1600

Raman shift (cm^{- 1})

FIGURE 2. Deep-ultraviolet resonance Raman (DUVRR) spectroscopy can be used to characterize lysozyme, a well-known protein which readily forms fibrils under certain conditions. Experimental (blue), modeled (red) DUVRR spectra and their difference (green) obtained for the lysozyme incubated for various times show changes in the protein structure at the early stage of fibrillation.

Applications

1-866-jobinyvon
www.jobinyvon.com

Complete Systems

Visit us at Photonics West, Booth #621

cule. The amide chromophore Raman signature provides direct quantitative information about the secondary structure of proteins. Vibrational bands of aromatic amino acids, tryptophan and tyrosine, have been shown to be responsive to contacts between secondary structure elements and exposure to water and, consequently, are used for characterizing the tertiary structure of proteins.

A new deep-UV Raman spectroscopic apparatus utilizing a laser source tunable between 193 and

205 nm was specifically designed for DUVRR protein structural characterization (see Fig. 1). ⁸ A 197 nm, 1 mW Indigo-S laser system from Coherent (Santa Clara, CA) is focused into a spinning nuclear magnetic resonance (NMR) Suprasil glass tube containing 150 μ L of solution. Scattered radiation