Methodologies in Gas-Phase Spectroscopy for Study of Polypeptides

The secondary structures of proteins, such as α-helices, β-sheets, or β- or γ-turns, mainly determine the overall 3-dimensional structures of protein and these structures are mainly stabilised by interactions such as hydrogen bonding (H-bonding) and dispersion interactions. Besides that, the interaction with its biological environment also contributes to the overall structure which means the intrinsic properties of the protein are hidden. Therefore, to have a better understanding about protein structure, proteins or polypeptides must be in isolated condition where no solvent molecules are present. One of the methods that can be used to study peptides in this isolated is gas-phase spectroscopy.

3D structure of myoglobin with α-helices and random coils are shown

Gas-phase spectroscopy can provide information about intrinsic properties of the building block of polypeptide which is commonly hidden in its surrounding. Besides that, from this spectroscopy the strength of interactions within the polypeptide that give the 3D structure can also be observed. One of the advantages of gas-phase spectroscopy is it can give the best data for comparison with theoretical calculations. Furthermore, the theoretical calculation will be commonly used to account for the spectrum of the polypeptide.

Optimised minimum energy structure of Phe-Gly-Gly. Calculated frequencies are shown as stick spectra and IR-UV SHB spectra.

Despite the combination of gas-phase spectroscopy and theoretical calculations, the study in the gas-phase spectroscopy of polypeptides emphasises the probing of molecular interaction rather than determining the structure.

This technique implies that the polypeptide needs to be brought into gas phase to study the electronic spectrum of the polypeptide. However, there are some problems to vaporise the polypeptide in the experimental conditions, which are low vapour pressure of polypeptide and decomposition under extreme heat. Luckily, there are many techniques that are commonly used to bring this type of molecule to the gas phase but these techniques are mainly used in mass spectrometry. One of the methods that can be used to bring polypeptide into gas phase is laser-desorption jet cooling method.

In this method, the sample is mixed with graphite powder and applied to a solid bar such as graphite and then laser brings the sample, in this case polypeptide, to gas phase by desorbing from the mixture and cool it down by supersonic expansion with the noble gas such as He or Ar as the carrier gas. Furthermore, the common laser source in this technique is Nd:YAG laser (1064 nm). Another method to produce gas phase polypeptide is by using nanoelectrospray from an acidic solution of volatile solvents and the molecule is trapped using multipole ion trap. This method is commonly used to study the isolated protonated peptides which have overall positive charge.

As mentioned earlier, this spectroscopy is electronic spectroscopy and types of electronic spectroscopy in the study of peptides are double-resonance spectroscopy such as resonant 2-photons ionisation (R2PI), IR-UV or UV-UV spectroscopy. These methods are used to explore different energy level of the molecules using electromagnetic radiation as shown below.

How the energy levels can be explored using electronic spectroscopy techniques.

With the ionisation process in R2PI technique, it has advantage of mass selectivity. Meanwhile in UV-UV or IR-UV, one of the pulses excites the molecule so the ground state is depleted and the other pulse follows the first pulse as a probe with a time gap between 50-100 ms. Because the first beam causes a depletion of the ground state, it is called a burn laser pulse, and due to this depletion a signal decrease of the probe laser pulse which cause a 'hole' in the spectrum; hence UV-UV or IR-UV spectroscopy is known as spectral-hole burning (SHB). In Ir-UV technique, IR pulses act as the burn laser pulse to excite vibrational transition which gives the advantage of this technique to be isomer selective and also can be used in structural assignment.

Another method to probe the conformation of small peptides or amino acids can be done by examining X-ray photoemission (XPS) and near edge X-ray absorption fine structure (NEXAFS) spectra. These methods probe electronic transition within the molecule and the peak-shifting could predict the interaction within the peptides.

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