Top-down Proteomics

Analysis of Cultural Heritage samples using top-down proteomics.


This project aims to harness the potential of top-down proteomics and obtain new valuable information on the degradation mechanisms of the organic media (e.g. protein breakdown) and protein chemical modifications such as oxidation, deamidation etc. (i.e. impact of restoration procedures and conservation conditions at molecular level) previously not accessible by conventional bottom-up methods.

What is top-down proteomics?

Top-down proteomics analyses intact proteins without digestion providing a global and comprehensive analysis of proteoforms – all protein forms from a single gene due to genetic variations, post-translational modifications, alternative splicing . Intact proteins are injected into the MS instrument, and later fragmented using the wide range of fragmentation tools available, to give an in-depth sequence information. As this approach gives information about both the mass of the intact protein and the structural information based on the fragments recorded, It offers a “bird’s-eye” view of the proteome allowing to decipher post-translational modification codes, genetic mutations, and alternative splicing.

By analysing the protein in its intact form, we can obtain additional information on the structures and relative abundancies of different proteoforms that often are not possible to well characterise using bottom up proteomics

Why top-down?

The most common proteomics approach to the identification of protein compounds has been bottom-up proteomics, a technique where proteins are extracted from a sample and are digested into peptides using enzymes. This digestion step can often lead to introduction (i.e. deamination), loss, or degradation (i.e. phosphorylation) of protein modifications due to associated conditions, which in turn limits the information about these modifications that can be obtained. Furthermore, in this approach each identified peptide acts as a sign for the presence of the protein molecule from which it is derived, but as multiple isoforms and proteoforms can contain the same peptide, direct information about them is lost. Therefore, information can only be inferred in regards to the actual proteoforms from which the peptides were derived. In the alternative top-down approach these issues are circumvented by analysis of whole proteins.

A key part of our experiment procedure is making paint models that we can use to optimise our analysis. This involves innovative and minimally invasive sample preparation methods for proteins extraction from their matrix, without induced modifications and breakdowns. It involves as well the development of whole miniaturized workflows and optimal instrumental settings, as well as adapted data processing methods. Here, you can see two spectra showing the lysozyme protein that is found in egg white. Spectrum A shows a lead white paint model made using a pure lysozyme laboratory standard, while Spectrum B shows a lead white paint model using egg white. By looking at the full range of charge states present, one can see the substantial change in the complexity of the protein.

Due to the high degree of protein chemical heterogeneity of these spectra the assignment of observed peaks is often highly challenging, which places emphasis on the development of optimised methods for high resolution data acquisition, as well as development of computational tools for their analysis.

Further reading

Smith LM, Kelleher NL. Proteoforms as the next proteomics currency. Science. 2018;359(6380):1106-1107. doi:10.1126/science.aat1884

Research performed in collaboration with…