|Earlier this year Methods published an article entitled Using Hydroxyl Radical Footprinting to Explore the Free Energy Landscape of Protein Folding. The article provides guidelines to ensure best practises when protein mapping using the emerging technology called fast photochemical oxidation of proteins (FPOP). Recently, BSI spoke with lead author, Dr. Antonio Calabrese from the University of Leeds to discover more about his research.Understanding structure, flexibility and dynamics of side chains in different protein conformers is critical to structural biologists. While very successful for other applications, nuclear magnetic resonance (NMR) is limited in its ability to perform direct comparison on partially folded proteins due to conformational exchange; thus other methods are required.|
University of Leeds
FPOP is a novel mass spectrometry based method for protein footprinting that has the ability to characterize protein variants in different conformational states. This is achieved by determining conformational changes in proteins caused by folding/ unfolding, with the added capability of investigating changes in solvent accessibility that accompany ligand binding. Further, the speed of FPOP avoids conformational averaging. According to Dr. Calabrese:
The lab is very interested in method development, developing new techniques and applying a variety of mass spectrometry technique to study protein structure. We also work with native mass spectrometry and chemical cross-linking and so… labeling with hydroxyl radicals is complementary to hydrogen deuterium exchange (HDX). One of the things different about it is because you have a covalent label you can be a bit more rigorous with your downstream sample handlings.
Within the realm of protein mapping, a significant interest of the group is surface mapping. To accomplish this, the surface area of a protein is labelled with hydroxyl radicals; opening the opportunity to identify labelling sites at the amino acid level and perform quantification on the observed oxidation.
|Dr. Calabrese was not shy about addressing the challenges of FPOP:
The thing that is rather complex about the data that you get out of FPOP is that generally speaking you are looking at modifications which are quite lowly abundant and so being able to reliably quantify those levels of oxidation and quantify those modifications is often quite challenging; especially if you want to analyze your data in order to obtain very specific information…. We used PEAKS to help assign our modification sites and then we also had to manually validate, specifically the modification, sites.
Peptide level quantification of oxidation levels.
In addition to using PEAKS for identification, the quantification node, PEAKS Q, was applied in both label and label free techniques. Precursor Ion Quantification (traditionally used for ICAT/SILAC) was configured for custom variable modification mining and Label Free Quantification for the remainder of features. When asked about satisfaction from the automated results produced by PEAKS the answer was very positive:
The automated results were definitely quite good. Whenever we manually validated those automated results they were pretty much spot on. When results are right it is obviously much easier to work with. Also, the capacity to look at the de novo spectra means you have got a lot more data to play with; otherwise much of it just gets thrown away. […] Now we are using it routinely for all of our DDA data analysis in the lab.
As with many other emerging technologies, the project is set to progress onto other complicated systems where it can continue to be complementary to HDX.
We have just looked at some relatively simple systems [soluble proteins], but now we are starting to look at more complicated systems using the techniques; so now are starting to look at characterizing membrane proteins which are obviously a lot more challenging and trying to optimize everything now.
We are grateful to have had the opportunity to speak with Dr. Antonio Calabrese and receive insight into emerging methods that dig deeper into explaining our proteomic world.
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