Next-generation proteomics for the Cambridge Biomedical Research community.

It is now clear that much of the complexity afforded by our biological machinery is at the level of protein variation rather than due to a high number of distinct genes. The divergences among highly related, but chemically different, protein molecules arise from allelic DNA variation, alternative splicing and chemical modification of the proteins once they have been synthesised. This generates a vast array of protein variants each having subtle but specific cellular roles. Each has differences in mass and can therefore be studied by using a mass spectrometer giving further insight into disease mechanisms.

Mass spectrometers measure the mass of charged molecules (ions) such as proteins and peptides. From these mass measurements, both the identity of proteins present and their amount can be calculated. This has applications across virtually all aspects of human cell biology and physiology in health and disease. In addition to knowing which proteins are present in their samples, researchers also want to know how abundant they are and how their abundance changes in response to stress, drug treatment or disease state. Also, they often want to know how proteins are regulated via chemical modifications to specific amino acid residues in their sequence (post translational modifications or PTM's) and how these change upon perturbation. The increasing sensitivity and resolution of modern mass spectrometry instrumentation is now allowing an unprecedented characterisation of proteins within complex biological samples.
The CRUK Cambridge Institute Proteomics Facility (CIPF) is a well-established internationally recognized proteomic facility offering access to proteomics expertise and infrastructure primarily to the Cambridge biomedical community but also nationally and internationally. CIPF uses mass spectrometry as its major tool to interrogate the proteome and in this application seeks funds to extend its capabilities through the purchase of a state-of-the-art instrument, the Orbitrap Eclipse Tribrid Mass Spectrometer with an associated uHPLC. The Eclipse is much more sensitive than CIPF's current instruments. It processes samples more quickly and hence will increase the capacity of CIPF and reduce sample turnaround significantly. This instrument excels at quantitative measurements using isobaric tagging methods. Its extended mass range opens the way to analyse protein isoform (proteoforms) and its ion mobility function helps distinguish biomolecules with similar masses but subtly different behaviors. Together this instrument will allow proteomic analysis of the most challenging low-abundance or highly complex biological samples, identify more proteins and their PTM's and quantify their levels more accurately.

The cohort of co-applicants in this proposal are world class scientists from diverse parts of the Cambridge biomedical research community and their research includes stem cell research; epigenetics; infectious diseases; mitochondrial biology, critical care (acute respiratory disease) and cancer biology. To help them achieve their research aims they require more advanced workflows to gain greater depth of information and with a higher degree of confidence. Increasingly requests are received for analysis from samples where cell numbers are limited and/or where the more elaborate analysis of ultra-low abundant peptides is required. The acquisition of the Eclipse importantly will provide added sensitivity and functionalities that will be transformational to the MRC-funded research detailed here, our current user base and the wider Cambridge biomedical community.

CIPF request funds for an Orbitrap Eclipse Tribrid MS coupled to a Vanquish Neo UHPLC. The Eclipse has a quadrupole mass filter and two mass analysers which can both operate sequentially or in parallel at acquisition rates up to 40Hz, twice as fast as current MS platforms at CIPF. It will increase throughput and depth of analysis required for the analysis of samples from rare and/or limited cell types, for example, and will have far reaching impact on understanding infectious disease models (Conway Morris, Summers), embryogenesis (Kelsey) and patient primary cells in the context of haematological malignancies (Huntly). The speed and sensitivity results from new segmented quadrupole mass filter, which in combination with Real Time Search capability and resolution of the ultra high-field Orbitrap, provides more accurate TMT-based quantification and more peptide identifications per protein (crucial for all applicants). These are also important improvements for the identification/quantification of low abundant peptides such as post-translationally modified peptides that lead to mechanistic insight in neutrophil (Conway Morris), and RNA biology (Hannon, Lilley), transcriptional regulation of DDR and oncogenesis (Jackson, Carroll, Huntly), oxidative damage response (Murphy) cardiovascular and respiratory diseases (Conway Morris, Summers). Finally, the extended mass range and PTCR function allows the analysis of large multiply modified peptide ions/intact proteins that will identify biologically relevant structural information of protein isoforms (Jackson Hannon, Kelsey). Overall, the flexibility of the Eclipse will provide a range of analytical service (D'Santos, Papachristou) to several outstanding MRC-funded groups in Cambridge who are world leaders in their fields and will contribute to the success of their projects addressing important biomedical questions.