Gas-phase structure of deprotonated amino acids identified by FELIX

31 March 2009
Although biologically relevant molecules are widely studied by mass spectrometry, many fundamental questions regarding their gas-phase structure remain, even for the simplest bio-active molecules. Using the free electron laser FELIX, Jos Oomens and coworkers at the FOM Institute for Plasma Physics Rijnhuizen have identified the deprotonation site of a series of gas-phase amino acids. In general, knowledge of the precise structure of biomolecules is key to understanding their functioning.

The 3-dimensional structure of biomolecules is largely determined by electrostatic interactions within the molecule and with the environment. Depending on the pH of the environment, acidic residues in proteins are often deprotonated and basic residues are protonated, so that positively and negatively charged sites are formed, which strongly influence the molecular structure. Virtually all proteins possess multiple acidic and basic residues and which of those become (de)protonated depends on the site-specific basicities and acidities. Even for some of the simplest biomolecules (such as amino acids) in complete isolation (in the gas phase), this question remains under debate. Apart from the backbone amino and carboxylic acid groups, an amino acid possesses a side chain, which can be neutral, basic or acidic.

It has been assumed that gas-phase amino acids deprotonate uniformly on the backbone carboxylic acid, forming a carboxylate anion. However, this has recently been cast in doubt for cysteine and tyrosine, which have been suggested to deprotonate on the side chain forming a thiolate and a phenoxide anion, respectively.

Well-known methods for structural determination of biomolecules, such as NMR, x-ray crystallography and IR absorption spectroscopy, are not applicable to the extremely low-density gas-phase ion cloud in a mass spectrometer. However, FELIX can be applied to record an IR photodissociation spectrum, which can be compared to calculated IR spectra to identify the molecular structure.

IR photodissociation spectra have thus been obtained for a series of deprotonated amino acid anions, including cysteine and tyrosine. All spectra match with spectra computed for the carboxylate form of the anion. Deprotonation on the side chain can be excluded on the basis of these spectra (see Figure for tyrosine).

Results of this study have recently been published in the Journal of the American Chemical Society, see http://dx.doi.org/10.1021/ja807615v.