Probing biomolecular interactions by combining ETD-tandem mass spectrometry with chemical footprinting methodologies.

In life, most proteins function as an integral part of a biomolecular complex consisting of proteins, nucleic acids, small molecules and metal ions, all held together in a specific 3D architecture. Correctly functioning proteins maintain health in plants and animals, the latter including humans. However, protein mis-function can lead to disease: for example, unwanted protein self-aggregation can produce insoluble amyloid plaques which are associated with well-known diseases such as Alzheimer's, Parkinson's and Type II diabetes.

We propose to purchase a state-of-the-art mass spectrometer which will enable us to pin-point protein-protein and protein-ligand binding sites. Our strategy involves subjecting a protein, or protein complex, which is in its native (3D) conformation, to chemical modification (or "footprinting") techniques. The regions of the protein that will be modified chemically are those which are exposed on the exterior of its structure, whilst the regions lying in the interior of the structure are inaccessible and remain unmodified.

In order to identify the sites of chemical modification, we need to use advanced mass spectrometric techniques that will tell us not only the molecular mass of the protein, but also its amino acid sequence. During protein sequencing, it is apparent which amino acids have been modified due to the change in the mass of the residue due to the chemical modification. For example, if the amino acid is oxidised, it will gain an oxygen atom and a corresponding increase of 16 Da.

Our proposed combination of advanced mass spectrometry with chemical modification methods will provide new information concerning:
a. the 3D structure of the protein by disclosing which regions are on the surface and which are in the interior of the structure;
b. protein-protein and protein-ligand binding sites, by comparing the "footprint" of the protein alone with that of the protein bound to a small molecule or another protein, we can discern where the ligand is bound;
c. protein function. We can monitor the protein's chemical footprint over time to map any structural changes occurring e.g. unfolding, binding, self-aggregation. This will provide key insights into how proteins function and mis-function;
d. the design of potential therapeutics. To prevent a protein from mis-functioning, it is often possible to add a small molecule drug to prevent an unwanted event e.g. to prevent a protein from unfolding followed by self-aggregation, it is possible to add a small molecule which will compete successfully for the protein-protein binding site. Our methods will locate the binding site.

The beneficiaries of these proposed studies will be the bio-scientists who will gain a significantly improved understanding of the ways in which proteins and other biomolecules function and mis-function. This will lead to improved drug design by pharmaceutical and biopharma companies, which in turn will lead to a healthier life for all of us.

In vivo protein-protein and protein-ligand interactions are vital for proteins to function, as most proteins operate in non-covalently bound biomolecular complexes. Thus it is necessary to map protein-protein interactions binding sites to understand how proteins function and mis-function.
We wish to purchase an ETD-MS/MS mass spectrometer to use in combination with an in-house established range of chemical footprinting methodologies to characterise biomolecular interactions and associated structural changes at the amino acid level.
Using chemical cross-linking (CCX) methods plus fast photochemical oxidation of proteins (FPOP), we can selectively modify, or label, exposed amino acid residues in proteins and biomolecular complexes. Thus we can determine amino acid distance constraints (using CCX) as well as distinguish exposed residues from unexposed residues, which are shielded within the biomolecule's 3D structure and remain unmodified (FPOP).
Determination of the location of the chemical modifications depends on the mass of the protein. If below 30,000 Da, then ETD-MS/MS can be used to sequence the intact protein and locate which residues have been modified following FPOP. If the mass is 30,000+ Da, proteolysis is used to generate smaller peptide fragments following CCX or FPOP. However, these peptides are often still too large for CID-MS/MS sequencing (max. ~5000 Da) and so also require ETD-MS/MS sequencing.
Currently we have only CID-MS/MS and thus we are requesting the complementary ETD-MS/MS to underpin and support much of our funded bioscience research.
Further, for protein-ligand binding studies which are crucial for determining how undesirable protein aggregation can be inhibited and for development of a plethora of therapeutics, ETD-MS/MS is necessary to sequence the protein while maintaining the non-covalently bound ligand attached. CID-MS/MS dissociates non-covalently bound complexes and so all information concerning the ligand binding site is lost.

Scientific impact:
Researchers studying a wide range of structural molecular biology enigmas, including protein folding, aggregation and biomolecular complex assembly in the fields of virology, membrane proteins, protein mis-folding diseases and protein/small molecule therapeutic design will all benefit. Our multi-disciplinary, funded research has both biochemical and medical significance and is of undoubted importance for health and well-being. The technology developments proposed in this application will be of interest to a wide spectrum of scientists, pharmacists and medical researchers in both academia and industry.

Industrial impact:
The Astbury Centre for Structural Molecular Biology (ACSMB) has many long-standing industrial collaborations which culminated in in the establishment of the Pharmaceutical and Biopharmaceutical Innovation Hub in 2013.

In particular, the PI and CoIs have many strong industrial collaborations ranging from pharmaceutical and biopharma companies (AZ, GSK, iQur, Medimmune, UCB) to instrument manufacturers (Avacta, Waters) and including the UK National Measurement Institute for Chemical and Bioanalytical measurements (LGC). Funded BBSRC and EPSRC postgraduate CASE PhD studentships and post-doctoral fellows, plus donations of instrumentation (including over the past 15 years second-hand mass spectrometers from AZ, Pfizer, Syngenta, and Waters) confirm the continued interest of Industrial support for the ACSMB in general, and the Biomolecular Mass Spectrometry Facility in particular.

Supporting knowledge and technology translation:
The ACSMB hosts an array of seminars, research themed workshops e.g. "Imaging and Single Molecule Techniques", "Protein Modification Technologies" and "Biomembranes", and public lectures (e.g. celebrating the Braggs' centenary), in addition to its annual retreat. The three Faculties involved: Biological Sciences, Physical Sciences and Medicine & Health all host annual postgraduate symposia in which all students are expected to present.

The PI and CoIs present research regularly at major national and international scientific conferences and have won many distinguished awards, including recently: the 2009 ASMS Ron Hites prize for "outstanding research" (Ashcroft and Radford); the 2012 Supramolecular Chemistry RSC Bob Hay lectureship (Wilson); the 2014 Potts Medal Award for Outstanding Contributions to Chemistry from Liverpool University (Ashcroft); the 2013 RSC Carbohydrate Chemistry award (Turnbull) and the 2013 Protein Society Carl Branden Award (Radford).

Delivering highly skilled people:
We will provide hands-on training for PhD students involved directly in mass spectrometry projects, and add lectures and course-work detailing our new chemical footprinting and ETD-MS/MS analyses to our annual PhD student/post-doctoral fellow MS course. External students can attend the course if relevant. Thus, our students will be trained in cutting-edge technologies and develop new skills in advanced methods of structural molecular biology and mass spectrometry. This experience will benefit their future careers, whether in academia or industry. Over past years, our highly skilled PhD students and post-doctoral fellows have gone on to post-doctoral fellowships and lectureships in other universities, and positions in many industries.

Public engagement:
The ACSMB's members have a strong track record of engagement with the general public (e.g. the Bragg centenary lectures, Café Scientifique), including science fairs to inspire school children to study science, student placements in research laboratories, talks at conferences for school teachers and in schools, and authorship of articles for sixth-formers. ACSMB works closely with CampusPR to promote its science effectively to audiences outside academia, in print (newspapers, popular scientific journals), on-line (e.g. BBC, Daily Telegraph, industry-focused websites) and on the radio.