MJ Dunn Fellowships
Dr Maruan Hijazi (QMUL)
My name is Dr Maruan Hijazi. I have been awarded with a MJ Dunn Fellowship to attend the annual BSPR meeting 2019 at Southampton. It has been a pleasure to attend this scientific meeting with leaders in the field of proteomics across the UK and Ireland. This year, the open plenary talk was given by Mike Snyder from Stanford University. Previously, there was a training workshop tutored by Luis Mendoza, from Seattle. The programme covers new developments in proteomics, state-of-the-art techniques and emerging advances. There are other talks that complement omics technologies (genomics, transcriptomics, metabolomics, etc) for personalised medicine, disease diagnosis, drug development and novel biomarkers as well as the analysis and integration of proteomic data.
I highly recommend more students and postdocs to join this fantastic society. It is a wonderful opportunity to meet other proteomics groups and stablish a personal networking (e.g. the wine reception and the conference dinner served to engage with colleagues in an informal environment). This annual event provides the perfect forum for the presentation of key achievements in proteomics to date. I was invited to give a talk where I showed our databases that represent a unique resource to investigate the relationships between kinase network topology and cell phenotypes. Finally, I would like to thanks the conference organisers and promoters who made the meeting possible. Their work to organise and coordinate the scientific programme has been excellent. Hope to see you all next year in Oxford (BSPR 2020).
Dr Harvey Johnston (UCL)
No report provided
Georgina Charlton (University of Warwick)
Report Abstract: Methyl viologen is the main ingredient in paraquat, a wide spectrum herbicide used for weed control, and kills plants by causing oxidative stress. As a result of methyl viologen action the production of antioxidants decreases, and the production of reactive oxygen species increases, leading to oxidative damage. Oxidative damage causes irreversible damage to proteins, leading to loss or change in function. Carbonylation modifications have been established as a key biomarker of oxidative stress, however, due to their low abundance they have proved difficult to analyse in the past. To analyse carbonyl modifications effectively an enrichment method is needed, however many current methods are focused on the protein level, meaning peptides identified by mass spectrometry aren’t necessarily modified, leading to loss in sensitivity of site-specific information. In this work we aimed to develop an effective enrichment method for carbonylated peptides to identify sites most susceptible to carbonylation. Work began on the single protein BSA to develop an effective method, and then progressed to Arabidopsis, as a more complicated system. In the future we intend to move onto maize and attempt to find a link between drought stress and oxidative damage.
Report: Oxidative damage is caused by reactive oxygen species (ROS) to mediate cell signalling pathways. Cells have antioxidants to keep levels of ROS under control and keep oxidation under control. However, under oxidative stress conditions levels of ROS are too high for antioxidants to control, meaning oxidation happens uncontrollably, leading to protein damage which can cause loss or gain in function. Carbonylation is a relatively stable form of oxidative damage which has been identified as a key biomarker for oxidative stress. Due to the low number of these modifications they have proved difficult to analyse, so an effective enrichment strategy is needed to fully understand these modifications and how they affect proteins.
Methyl viologen (MV) is the main component of paraquat, a wide spectrum herbicide produced by Syngenta, and is known to induce oxidative damage. Methyl viologen attacks plants at photosystem one during photosynthesis, inhibiting the ferredoxin reaction, NADPH regeneration and ATP generation. In this work methyl viologen was used to induce oxidative damage in Arabidopsis thaliana to test a method for tagging and enrichment of carbonyl modifications. Soluble and membrane fractions were isolated for separate analysis.
Proteins were quantified using the Tandem Mass Tag (TMT) 10plex, proteomics was performed on an LC/MS orbitrap fusion and data analysed using MaxQuant. TMT 10plex allows for direct quantification between samples. Samples can be tagged with different TMT 10plex molecules and then combined for analysis. Each tag has the same isobaric mass and chemical structure, however the placing of various heavy isotopes at different positions within the molecule allows for different masses when the fragmentation during MS2 occurs. The mass reporter ion which will be different for each sample is then able to be used to quantitatively measure protein expression during peptide fragmentation.
In total 1336 proteins were identified in the soluble fraction, of which 220 proteins were statistically significantly less abundant and 164 proteins were statistically significantly more abundant in the MV treated compared to the control. Of the less abundant proteins they were mostly chloroplast metabolism proteins, and the more abundant proteins were mostly stress response proteins. In the membrane fraction 879 proteins were identified, of which 386 proteins were statistically significantly less abundant and 231 were statistically significantly more abundant in the MV treated compared to the control. We established that methyl viologen was changing the proteins and causing damage, we could therefore carry on with testing tagging and enrichment for carbonylation.
To enrich for carbonylation we have been using alkoxyamine-peg4-biotin and monomeric avidin beads. An enrichment can be performed at the protein level, with an on-bead digest, this will give information on the proteins which are carbonylated, but also give a lot of background peptides which are not carbonylated. Enrichment at the peptide level, with elution form beads will give information on the sights which are carbonylated, but this is more difficult to achieve due to the strength of the biotin avidin interaction.
We have been able to establish a model sample which is oxidatively damaged and have been testing tagging and enrichment strategies both at the protein level and the peptide level. Once an effective method is established, we intend to move to testing for carbonylation in droughted maize samples, as we believe there is a link between drought stress, carbonylation and yield loss. Maize is the worlds third largest crop for food production, and with a growing global population, the need to feed more people is increasing. An insight into yield loss at the molecular level is vital to our ability to prevent crop loss in the future.
What the conference meant to me: ASMS is the largest mass spectrometry conference in the world. I have never been to a conference this large before. Having several parallel sessions and nearly 800 posters per day was overwhelming, but it was great to see all the work going on in the field of mass spectrometry and new innovations. But there was always something to do weather it was listen tom talks, talk to vendors or look at posters. I was able to talk to people from all over the world who were working on similar things to me and we were able to discuss techniques and ideas for going forward within our work.
No reports provided
James Waddington (University of Liverpool)
Maximilian Harris (University of Liverpool)
Augustine C. Amakiri (University of Liverpool)
Grigorios Koulouras (Beatson Institute)
Kareena Adair (University of Liverpool)