Proteomics Facility

Proteomics Facility

As part of the National Phenome Centre (NPC) at Imperial College London, we are incorporating proteomics to complement our existing lipidomics, metabolomics and genomics technology. As we develop multi-omic methods, it is a great time to discuss any proteomics that you may want to perform now or in the future. To find out more about what we are working on or to get in touch, read below!

Proteomics

Proteomics is the study of proteins. Proteins are one of the major molecular classes in the body, and are critical for controlling chemical reactions, allowing us to move, sense, taste, breath...everything! There are approximately 22,000 genes in the human genome that each contain precise instructions to make different proteins. But there are well over 1,000,000 different proteoforms in the body as they can be extensively modified to change their location, function and lifespan (turnover). Proteomics is the study of all these, to find out their biological purpose and how they perform their precise functions in health and disease. Within this facility, we focus on clinical proteomics and are developing methods for high-throughput serum proteomics, multi-omic analysis (metabolomics and genomics), targeted quantitative proteomics as well as whole proteome profiling.

Mass Spectrometry

Mass spectrometry (MS) is the principle tool of proteomics, it is an incredibly powerful technique for identifying and quantitating millions of different proteins/proteoforms. Mass spectrometry works through determining the mass of a molecule, with each molecule having a specific mass or series of masses. It can be coupled with chromatography (LC-MS) to give very accurate quantification. There are many mass spectrometry methods adjusting speed, resolution, and accuracy to the experimental requirements. MS can be set up to detect either a single protein/peptide, or thousands of different proteins in a single run.

Mass Specomtrometry

Proteomic Profiling

Synapt G2S

Whole proteome profiling of cell lysates, tissue or bio-fluids, with or without sample pre-fractionation can be performed on our nanoLC (Acquity, Waters) coupled to our IM-QToF instrument (Synapt G2S Waters). This system features ion mobility to further reduce sample complexity and increase proteomic coverage. Acquisition can be through data-dependant (DDA) or data independent (DIA) acquisition approaches. DIA is performed through MSE, this approach is truly data independent, both at the data acquisition and database searching stages. Quantitation can be performed by the Top3 approach using internal standards.

Mass Specomtrometry

High Through-put Serum Proteomics

Xevo XS

We are setting up methods for high-throughput serum proteomics with and without depletion. SONAR acquisition is a DIA approach whereby a quadrupole scans across a mass range, to reduce the complexity of ions reaching the TOF during MS1 and MS2 acquisition. This approach combines the best of DDA (i.e. clearer fragmentation spectra) with the quantitative capabilities of DIA. We are implementing this technique on our Xevo G2S with microLC to enable rapid and reproducible LC separation of peptides. This technique is perfect for the analysis of serum, generating quantitation of ~200 proteins without pre-depletion or fractionation.

[Data in heatmap from Hughes, King and Gethings (2019) and analysed in house with DIA-NN (Demichev 2019)]

Mass Specomtrometry

Metabo-Proteomics

The NPC has established workflows for metabolomics. To combine this with proteomics, we are developing methods for the simultaneous preparation of metabolites and proteins for quantitative analysis. This will give in depth information regarding changes to molecular pathways, mediated by proteins and metabolites within the cell. Of particular interest is the investigation of S-Adenosine-L-Methionine Cycle and Methionine Salvage Pathways.

Mass Specomtrometry

Proteogenomics

Proteogenomics combines transcriptomic and proteomic technologies to identify splicevarients, SNPs and novel-transcriptional start sights (e.g. trascribed lnRNA, novel peptides/proteins) that would be difficult with either technology alone. Working with the genomcis facility, we are implementing experimental and analytical pipelines for to exploit protein and transcript information to discovery workflows. As we expand this technology, we will develop in house spectral libraries to increase the sensitivity of detection of different splice-forms and SNPs for future experiments.

Targetted Proteomics

Targeted Proteomics

MRM Assay Development

The NPC has extensive expertise in targetted assay development. We are working with a number of research groups at Imperial College London to develop targeted assays for small signalling peptides in the blood. If you are interested in developing something for your own experimental requirements get in touch!