Two Dimensional Mass Spectrometry in a Linear Ion Trap

Cells, tissues, and organisms, including humans, are made of a variety of molecules, including proteins. Proteins are the 'molecular machines' responsible for everything a cell does, from replication to processing nutrients. A typical cell might have 10's of thousands of proteins doing myriad particular chemical 'jobs', including synthesizing or recycling other proteins, so the complement of proteins is constantly changing. The field of proteomics involves detecting as many of these proteins as possible, understanding how they change with time and in reaction to stress or chemical treatment. Knowing this information will allow bioscientists, from basic researchers to clinicians, to better understand what is occurring in the cell under conditions of malnutrition, disease, healthy aging, drought, or even pharmaceutical treatment. Developing better methods to measure the 'proteome' accurately and with sufficient detail is a key underpinning research goal which results in better science, better technology, and ultimately better health and healthier food.

We have recently made substantial advances in a novel technology, called 2-dimensional mass spectrometry, which offers the capability to sequence all peptide/protein components in a sample, rather than a select few. We have demonstrated the technology works in several publications and in preliminary data on a whole proteomic analysis herein. Additionally, we have demonstrated by ion trajectory modelling, that this 2-dimensional mass spectrometry method is not necessarily limited to the FTICR mass spectrometers, but can also be adapted to the less costly linear ion trap mass spectrometers. However, there are some particular challenges which we must address before the vision can be attained. This proposal aims to solve those challenges.

Firstly, we have built a prototype instrument but we must optimise, tune, and test it for proteomic samples. Secondly, we need to actually acquire data on those proteomic samples, using some standards such as yeast cells. Thirdly, we aim to explore new methodologies that might allow 3-dimensional mass spectrometry experiments. And, fourthly, we need to adapt the data from this 2DMS-LIT instrument to our data processing algorithms already in place. The overall result of this project will be the development of protocols and methods for optimal proteomic analysis using this exciting new technology and the ability to generate 2-dimensional mass spectrometry data for extraction of protein sequence information for whole cells and tissues, using a smaller, cheaper mass spectrometer.

We have recently developed 2-dimensional mass spectrometry (2DMS) on the FTICR mass spectrometer to the point where it can now be applied to complex mixtures of proteins and generate unbiased sequence information about all proteins/peptides in proteomics samples. Additionally, we have shown how to extend this knowledge to doing 2DMS experiments on the cheaper linear ion trap instruments which large potential savings in the expense of the instrument, expertise required, and speed. However, for this vision to be realised, a set of challenges must be solved, which is the goal of this proposal. This project aims to fund 1 postdoctoral research assistant (PDRA) at 100% FTE for 12 months to achieve these goals.

The test-sample to study for optimizing these methods will be yeast cells, and we will focus on developing the pulse sequences and fragmentation methodologies to get whole-cell yeast proteomic data using the prototype 2DMS-linear ion trap instrument shown in the proposal.

Specifically, we will:
1. align the UV and IR lasers to achieve good fragmentation for about 50% of the ions in a given scan
2. optimise the pulse sequences with standard proteins and peptides to achieve high quality 2DMS data for these 'clean' protein samples.
3. optimise and tune the experiment with yeast cells to generate full proteomic 2DMS spectral datasets using the 2DMS-LIT prototype.
4. Acquire and process 2DMS spectra for Yeast and other proteomic standards using the linear ion trap.
5. Develop pulse sequences for a 2DMS-2DMS experiment and explore the limitations of such a technique. Potentially, this technique could generate 2DMS spectra for every MS/MS fragment for every precursor, in one hyperdimensional data set.
6. Adapt our existing data processing algorithms to handle 2DMS-LIT data.

We have recently made substantial advances in a novel 2-dimensional mass spectrometry (2DMS) technology that can be applied in the area of proteomics, and have an ongoing BBSRC project to develop that technology on the FTICR mass spectrometer. We have also determined that it is possible to perform 2DMS experiments on a much less expensive and simpler instrument platform, the linear ion trap. This project is about transferring our knowledge of 2DMS proteomics experiments on the FTICR onto this prototype new linear ion trap instrument. Long term, the impact of this project will be in the following areas:

Economy
The proteomics market is substantial, with the annual market for proteomics equipment alone was >$10 billion in 2013 and is expected to reach >$20 billion by 2018. The total research and development effort using proteomics is far larger as these are fundamental tools that allow industrial and research scientists to detect proteins and protein changes in cells as a response to stimuli such as pharmaceutical treatment, disease, or drought. Fundamental new technologies, like those discussed herein, will have a large, long-term impact. New developments in mass spectrometry technology are likely to generate new intellectual property, which can be patented and licensed to commercial agencies such as Bruker UK.

People:
Economically, this instrument will be used to train PDRAs, PhD, MSc, and undergraduate project students in the methods of advanced mass spectrometry and proteomics. These trained personnel are a currently limiting resource for the UK bio-pharma, clinical, and medical industries, and they will have a large, long-term impact on that industry. The PDRAs will also be trained in science engagement with the public as part of their outreach activities described in the 'Pathways to Impact' plan.

Science:
Overall, the new instrumentation proposed herein will improve proteomics and other biomolecular analyses, which will improve data quality and analysis for bioscience samples throughout the biochemistry, pharmaceutical, medical, and clinical fields. The ability to know precisely which molecules are present and how they are modified will have a large impact scientifically.

Society:
More specifically, as deliverables for the 'Pathways to Impact Plan', this project will provide a data sharing website and a set of software packages and tools for researchers in this area, will provide new teaching materials in mass spectrometry related to the new instrument, and will generate an internet video of the experiment and technique to help train the next generation of students. Additionally, we will collaborate with our industrial partners to support CASE and other industrially funded PhD students to train those students on samples of interest to our partners, and thereby get those same partners interested and integrated into using these advanced mass spectrometry techniques.

Finally, the ultimate benefit of this research is that it will generate better instruments and methods to generate better proteomic data. This will lead on to better understanding of the underlying causes of aging, health, and disease. That understanding will lead to improved healthcare, diet, and disease treatment which will result in longer, healthier lives.