High sensitivity LC-MS to understand the role of Proteomes in the rules of life for Plant scientists and N8 partners

It has become apparent that biological complexity is largely orchestrated not through the number of genes but through variation at the protein level. Post-translational modifications (PTMs) generate different forms of a protein called proteoforms. These are responsible for cell signaling in almost every biological process. The dynamic nature of PTMs allows organisms to respond to highly transient changes in the environment with precise fine-tuning. This is especially relevant to plants due to their sessile nature; their growth and development is dependent upon their ability to adapt to changes in the environment. The reliance upon PTMs for plant responses to environmental change is orchestrated by thousands of genes encoding components of PTM systems and expansion in genes encoding the PTM machinery in plants is more significant than in any other kingdom. Understanding how these proteins affect cellular responses to environmental stress is a critical component of plant breeding programmes. This will be central in addressing the global challenge of sustainably producing food in a changing climate.
To date, a number of genetic variants which alter the PTM state of the corresponding protein have been found to be extremely valuable in agriculture. Examples are found in the DELLA genes, which result in a high-yielding variety of wheat that, due to the reduced height, is more resistant to damage from wind and rain. This valuable trait is controlled by a single mutant dwarfing DELLA, the rht-1 allele. The wheat rht1 allele produces a DELLA protein that is altered in its ubiquitinated state and acts as a dominant allele to increase resistance to adverse environmental stress, indicating that PTMs can have added value in expediting breeding programmes with dominant alleles. It is predicted that the critical difference between the flooding tolerant and intolerant SUB1A alleles in rice is the mutation of a phosphorylation site in the SUB1A protein, further highlighting the importance of PTMs in crop adaptation to the environment. Introgression of the flooding tolerant SUB1 allele is the single most significant advancement for generating flood-tolerant rice varieties in the last 30 years. PTMs can thus be exploited to generate novel alleles for boosting crop productivity. However, a systematic approach to exploit PTMs for plant improvement strategies has been limited by the lack of appropriate methodologies for target discovery, mass spectrometry machine access and training. With our track record in PTM analysis, especially in plants, we are proposing to bridge this gap by building a plant-cell-focused proteome research platform that will be used for method development and discovery of novel PTMs in crop species. This will add significantly to the huge tapestry of genomics data held by the UK and international model plant and crop communities and provide data for the design and implementation of future research and breeding programmes.
The acquisition of a highly sensitive mass spectrometer, such as the Bruker timsTOF Pro 2, with capability to detect these often-ephemeral modifications (as detailed in the proposal), will be central to our goal. The spectrometer will identify PTMs associated with beneficial traits while other technologies will elucidate the cellular responses to them. Although the focus is on plants the new mass spectrometer will equally serve a range of animal and microbial scientists in Durham who investigate PTM mediated signalling to understand the rules of life in various organisms. In some aspects PTM analysis in animal and microbial fields is further advanced than in plants and cross fertilisation of knowledge between these fields brings added value to this proposal.

Post-translational modifications (PTMs) underpin biological complexity through the generation of proteoforms. This is critical in multicellular organisms, where development requires signal integration to control and coordinate cell fate. Specific protein PTMs can occur in subsets of cells and subcellular compartments, limiting the number of modified target sites available for discovery-type proteomic analyses, and they are often transient, making it more difficult to isolate and identify them. Characterisation of PTM networks therefore requires sensitive analytical tools. The timsTOF Pro 2 mass spectrometer fits this purpose, with the capability of identifying high numbers of peptides from very low amounts of input peptide sample. This instrument acquires MS-MS spectra from peptides at >100 Hz, with parallel trapping and ion separation in a dual trapped ion mobility spectrometry (TIMS) cartridge. Synchronization of the TIMS device and quadrupole mass analyser reduces chimeric fragmentation spectra and TIMS reduces signal noise from contaminant ions. Parallel accumulation serial fragmentation (PASEF) technology enables the timsTOF platform to achieve increased depth of proteome coverage from reduced sample loadings. Additionally, the fast acquisition speed means that low-abundance precursors can be repeatedly re-targeted to improve MS/MS spectrum quality. These features are optimal for sensitive proteomic analytical capability at Durham, applicable to a range of strategic BBSRC target areas. Development of methods for PTM analysis, which will focus on plant material and isolated plant cell types is a key driver for the proposal. Output from these experiments will be integrated with transcriptomic, metabolomics, bio-imaging and modelling data for understanding of PTM codes. While the use of the timsTOF Pro 2 has a major focus on PTMs in plants, the capabilities of the instrument will equally apply to other projects outlined in the proposal.