Photodissociation of peptide model compounds
Mass spectrometry is an increasingly
important analytical technique for the characterisation of a range of
biological molecules. Structural information may be obtained using
tandem mass spectrometry (otherwise known as MS/MS) techniques, in which
a single species is mass-selected in the first stage of the
spectrometer, and is then fragmented and mass-analysed in the second
stage. This establishes the mass fragments associated with a given
molecule under the chosen fragmentation conditions, allowing structural
sub-units of the parent molecule to be identified.
Fig 1: Tandem mass spectrometry
Fragmentation studies on large molecules have
traditionally employed collisionally-induced dissociation (CID) or
infra-red multiphoton dissociation (IRMPD). In both of these
methods, energy is added to the molecule gradually – in the former case
through multiple collisions with an inert gas inside a collision cell,
and in the latter through sequential absorption of a large number of
infra-red photons – until it eventually reaches the dissociation
threshold. As a result, the fragmentation is a fairly slow
process, and the fragments are generally well thermalised.
Alternative fragmentation techniques include electron capture
dissociation (ECD) and ultra-violet photodissociation (UVPD), both of
which are much more ‘direct’ and rapid processes. There is
considerable current interest in UVPD from an analytical perspective, as
the fragmentation patterns observed are often complementary to those
seen in CID or IRMPD, and therefore provide further structural
information.
UVPD is often a very rapid process in
peptides [1], occurring on a much
faster timescale than intramolecular vibrational redistribution and
leading to ‘non-statistical’ fragmentation patterns (e.g. weaker bonds
may stay intact while stronger bonds are cleaved) and fragments with
non-statistical energy content. For example,
photodissociation at 157 nm has been shown to favour cleavage of a
carbon-carbon bond within the peptide backbone, rather than the
carbon-nitrogen bond favoured by CID and IRMPD [2],
and is thought to occur via a two-step mechanism involving an
odd-electron intermediate species, with side chain fragmentation also
occurring in some cases.
We are using new ‘multi-mass’ variants of the
velocity-map imaging technique, together with the H atom photofragment
translational spectroscopy technique (PTS), to better our understanding
of the UVPD process in peptides. Our studies are directed towards
understanding the nature of the participating electronic states,
together with the fragmentation mechanism(s) and dynamics of the
dissociation process. The first step is an investigation into the
dissociation dynamics of N,N-dimethylformamide, a useful small-molecule
model for the peptide bond in proteins, following laser photolysis at
157, 193, 248 and 266 nm. This compound is known to have a number
of accessible dissociation pathways [3],
which the velocity-map imaging technique will allow us to probe in
detail. Three previously identified pathways following 193 nm
photolysis are shown in Figure 1 below. Additional H-atom
elimination channels are highly likely, and these will be ideal
candidates for photofragment translational spectroscopy studies at
Once we have a solid understanding of some of
these ‘simplest’ of photodissociation processes in peptides, we will
broaden our studies to explore a range of di- and tri-peptides, in order
to investigate the effect of different amino acid residues on the
fragmentation mechanisms and dynamics. These investigations will
form the foundation for later studies on larger peptides.