We employ a number of characterisation techniques; most analysis is conducted in the gas phase, where we take care to gently introduce our samples from solution. We also use solution and solid phase analysis to complement our in vacuo findings

Ion Mobility Mass Spectrometry is our main technique – further information on this can be found here.

Nano Electrospray Ionisation – nESI

To be added

IM-MS and MS of Disordered Systems

The established approach to understand the function of a protein is to ‘solve’ its structure and subsequently investigate interactions between the protein and its binding partners. However, structure determination via crystallography or nuclear magnetic resonance (NMR) is challenging for proteins where localised regions or even their entire structure fail to fold into a 3D form. These so called intrinsically or inherently disordered proteins (IDPs) or intrinsically disordered regions (IDRs) constitute up to 40% of all expressed proteins, and a much higher percentage in proteins involved in the proliferation of cancer. Ion Mobility Mass Spectrometry is uniquely able to examine both absolute conformation(s), populations of conformation and also conformational change, and therefore is highly applicable to the study of IDPs.

Following n-ESI, all proteins are present in a number of different charge states, commonly as (multiply) protonated species (for positive ionisation) or (multiply) deprotonated (negative ionisation) – this is termed a charge-state distribution (CSD). Structured, folded proteins with little conformational flexibility in solutions where the protein is buffered appropriate to its pI, give rise to gaseous ions carrying a relatively low number of charges, presenting a narrow charge state distribution centered on high values of m/z. By contrast, less structured or unfolded proteins can accommodate a larger variation in the number of  protons related to the varied availability of ionisable sites on the protein surface, in turn a feature of the conformational flexibility in solution. Analysis of the relative intensities of ions in CSDs enables an indirect assessment of conformational families present in solution.

IM-MS provides another dimension to gas-phase studies of biomolecules garnering  more direct information on the molecular species ‘shape’ (collision cross-section, in Ų) along with the CSD, but does not provide atomic resolution compared to NMR and X-ray crystallography. IM-MS has been successfully applied to examine protein dynamics, protein-ligand and protein-protein interactions and shows a great promise for the structural characterisation of IDPs.

Molecular Modelling

In our group we utilize mainly Amber and NAMD software packages to carry out molecular dynamics (MD) simulations of peptides, proteins, protein assemblies and metal-binding proteins in vacuo and both implicit and explicit solvent.

Several techniques are implemented to satisfactorily enhance the sampling of the phase space, like for instance simulated annealing procedures and replica exchange MD, other than MD runs in canonical ensembles.

Electrospray ionisation (ESI) and, more specifically, ion mobility (IM) mass spectroscopy (MS) combined with molecular modelling comprise a very powerful combination of techniques for deriving and characterising molecular ions at the atomic resolution. After identifying the most abundant species (oligomer and related charge state) by nano-ESI-MS and obtaining the collision cross section through ion mobility, atomistically resolved structures can be determined by comparing the experimentally determined collision cross sections with those calculated for model structures. Accurate estimates of the orintationally averaged collision cross section for the complex geometries typical of all but the most simple molecular systems can be calculated using molecular methods and several levels of approximation. The most accurate scheme is termed the trajectory method which simulates multiple ion-gas collisions using an empirical potential for the ion-neutral interaction potential. Other methods are computationally much cheaper but rely on further approximations. The exact hard sphere scattering model still averages over a large number of collisions, taking into account multiple scattering events, but no interaction potential between the buffer gas and the ion is taken into account. The projection approximation equates the collision cross section with the orientationally averaged projection area of the molecule. The newly developed projection super-approximation also uses the projection area but rescales it in a fashion that emulates the long range effects, to achieve very good agreement between its estimates and those obtained using the aforementioned trajectory method.

The molecular modelling strategy can proceed in two distinct ways. First, if any other data is available, candidate geometries can be represented in silico and optimised using molecular mechanics techniques. Candidate structures with cross sections different to those obtained experimentally can be ruled out; in this way experimental data can be used to filter a set of molecular configurations. The second pathway for assigning structures to collision cross sections involves extensive sampling of the potential energy surface (using molecular mechanics methods as described above) to generate an ensemble of conformational models, which can be interpreted as geometries representative of the free energy basins of the molecule. The lowest-energy geometry from the set of molecular coordinates that are consistent with experiment can then be singled out as the most representative of the ensemble observed experimentally.

Other Structural Techniques (CD, TEM, High resolution MS, ECD)

In addition to ion mobility mass spectrometry we also utilise a host of other structural techniques. Including Fourier transform ion cyclotron mass spectrometry (FT ICR MS), which due to the high resolution of the data can enable us to distinguish between mass coincident species such as a monomers and dimers of twice the charge.  Also when coupled with electron capture dissociation (ECD) it allows us to probe peptide and protein sequence and structure. ECD is a method of MS/MS fragmentation by introduction of low energy electrons. Charge state reduction is the dominant process followed by fragmentation, during which cleavage occurs selectively along the backbone of the protein, primarily producing c and z –type fragments. In ECD bond dissociation following activation occurs much faster than typical bond vibration and hence fragmentation in this way is thought not perturb the higher order structure of proteins and peptides.

Other structural techniques used within the group include circular dichroism (CD) spectroscopy. CD is a low resolution technique which enables the secondary structure content of peptides to be probed. CD is the differential absorption of left and right circularly polarised light and occurs when a chromophore is chrial. In proteins, the distinct environments created by different types of secondary structure result in different CD signals. Through the coupling of the experimental data and various algorithms it is possible to gain an estimation of the secondary structure content present within our systems. Additionally, we often utilise microscopy techniques such as transmission electron microscopy (TEM). TEM involves the transmission of a beam of electrons through a very thin sample, in our case this is generally a peptide or protein solution applied to a TEM grid. The contrast depends on the atomic number of the atoms within the sample so for biological samples it is often necessary to stain the sample with a heavy atomic number stain, such as uranyl acetate. TEM is employed within our lab to gain insight into aggregating or amyloidgenic systems.

Protein Handling & Purification

We often either buy or samples or receive them from collaborating groups, however we sometimes express proteins and purify them ourselves. Proteins are expressed with the help of the Campopiano Group and grown on a large-scale in the School of Chemistry by John White.

We purify proteins in the Edinburgh Protein Purification Facility by various chromatographic methods such as size exclusion chromatography and ion exchange chromatography.

Our samples are generally sprayed from ammonium acetate buffer, in which case our proteins are dialysed into ammonium acetate using dialysis cassettes and any excess salt is removed by spin columns.