Raman spectroscopy was used to analyze two synthetic 21-mer peptides (F6 and L6) that differ in their amino acid sequences. Raman spectra of F6 powders showed peaks characteristic of phenylalanine aromatic groups, while L6 spectra did not show aromatic peaks. Raman spectroscopy can identify contaminants and characterize the secondary structure of peptide self-assemblies, and was shown to detect a downshift in F6 aromatic peaks after self-assembly indicating π-stacking interactions.

1. Peptides as biocompatible materials are ideal candidates for biological applications, given that their physical/chemical properties can be manipulated through rational design of their
amino acid sequences. Self-assembled fibril-forming peptides are a particularly interesting subset of this family, with the potential for novel functionalities. In this study, we analysed
two synthetic 21-mer peptides (F6 and L6) with similar amino acid sequences, with the distinction that one contains six aromatic phenylalanine groups per 21-mer peptide (F6) in
place of aliphatic leucine groups (L6). One tool that can be used to investigate the chemical properties of such fibrils is Raman spectroscopy.
Initial investigations focused on characterising contaminants. A characteristic absorbance in the UV-visible spectrum between 250 – 350 nm had suggested possible contamination
by fluorenylmethyloxycarbonyl (FMOC) derivatives since FMOC is used in the synthesis process. Raman spectra of lyophilised F6 powders (below) show peaks for the Phenylalanine
aromatic group at 1009 cm-1, 1038 cm-1, 1590 cm-1, and 1610 cm-1 [3]. On the other hand, given the absence of aromatic groups in the L6 Raman spectra, FMOC contamination was
eliminated as a possibility with the contamination remaining unidentified.
1009Aromaticvibrations
1038Aromaticvibrations
1345AmideBandIII(C-Nstretch)
1443AmideBandII(N-Hbend)
1610Aromaticvibrations
1661AmideBandI(C=Ostretch)
Aliphatic vibrations
3066Aromaticvibrations
CCDcts
Wavenumber (cm-1)
Raman spectra of L6 and F6 lyophilised powders
FMOC derivatives
L-Phenylalanine
Both L6 and F6 will self-assemble into fibrils within a known range of concentration and pH. Dried films of self-assembled F6 peptide were characterised by Raman as shown above
(right), indicating a downshift in the aromatic assigned peak compared with the unassembled F6 powder. This shift can be related to the π-stacking interaction of the phenylalanine
groups [5].
Peaks related to other chemicals in the peptide solution such as 2-(N-morpholino)ethanesulfonic acid (MES buffer) were assigned by comparing Raman spectra of powder and
solution forms and by comparison with reference spectra. The peak at 1050 cm-1 can be attributed to the sulfonic acid in the MES [6] was observed only for L6 dried film as shown
below (left).
Diphenylalanine (FF) and dileucine (LL) peptides were used as positive and negative references for spectral assignment of aromatic groups in peptides. Raman spectra of FF and LL
confirm the correct peak assignment of aromatic vibration bands in F6 powder spectra, providing spatial information also as the FF and LL nanotubes were visualised by raster
scanning a 100 x 100 μm area (below, centre).
Wavenumber (cm-1)
CCDcts
8 cm-1
1602
1610
Raman spectra of F6 lyophilised powder and dried film of self-assembled F6 solution
F6 powder
Self-assembled F6
π stacking
Overview of Raman spectroscopy [1,2]
CCDcts
Wavenumber (cm-1)
MES
Raman spectra of L6 lyophilised powder and dried film of self-assembled L6 solution
L6 powder
Self-assembled L6
1050
Raman spectroscopy in combination with Circular Dichroism (CD), NMR, and FTIR spectroscopy, enables the ready identification and approximate quantification of contaminants and
by-products in the commercially-synthesised 21-mer peptides. Moreover, it can be used to characterise the peptide self-assembly and resulting secondary structure formation, and
has the advantage of being a relatively simple analytical approach. Future experiments will focus on correlating shifts in Raman spectra to formation of peptide secondary structures
and time-series Raman spectra will be used during peptide gel formation to investigate the peak shifts due to π- stacking formation.
1. https://en.wikipedia.org/wiki/Raman_spectroscopy
2. Butler, H., et al., Using Raman spectroscopy to characterize biological materials. Nat. Protocols 2016, 11 (4), 664-687.
3. Lekprasert, B., et al., Nondestructive Raman and atomic force microscopy measurement of molecular structure for individual diphenylalanine nanotubes. Optics Letters, 2010. 35(24): p. 4193-4195.
4. https://en.wikipedia.org/wiki/Stacking_(chemistry)
5. Profit, A., et al., Evidence of π-stacking Interactions in the Self-Assembly of hIAPP(22–29). Proteins, 2013. 81(4): p. 690-703.
6. http://www.utsc.utoronto.ca/~traceslab/raman%20correlation%20table.pdf
[4]
LL FF
Raman
scan images
Raman
optical images
Raman spectra of LL and FF lyophilised powders
Wavenumber (cm-1)
CCDcts