 |
 |
|
 |
 |
 |
PARASITE PROTEOMICS
This area is the research interest of Dr J.V. Hamilton, Professor J. Barrett and Professor P. Brophy Additional information on these workers and their research can be found on the Group Members Main Page.
The parasitology group in Aberystwyth is actively involved in the use of proteomics to investigate various aspects of parasitology. The use of this technique for the study of various biological systems is becoming more widespread. This is due to the ever increasing volume of sequence information that is becoming available and improvements in our ability to both separate and identify individual proteins.
For those who are not familiar with proteomics, it is the investigation of the protein complement of an organism instead of the genetic material which is used in genomics. The genome of an organism is generally static, whereas the proteome is largely controlled by protein expression and can be changed by any number of factors. An organism will have a fixed number of genes (no doubt there are a few exceptions), a certain number of which will be expressed at a given time. Using proteomics we can look at the products of these genes and thus focus on the proteins that are actually present in that organism at the time of sampling. In this way we can investigate the response to different stimuli or compare, for instance, a disease condition to a control. Proteomics has a number of key advantages over other techniques such as microarrays that are also used for this kind of work. Using proteomics we look at the proteins present in the cell, not the mRNA from which it is translated. Thus we are looking at the true picture of what is happening inside an organism and not at what might be happening. Another fundamentally important advantage of proteomics is that it is possible to identify proteins that have been post-translationally modified. The modification of proteins plays a key role in the function of many proteins, therefore, our ability to look at and identify changes in these proteins can be of key importance. Many proteins are activated by phosphorylation, or are expressed in an inactive form (proproteins, preproteins or preproproteins) and must be cleaved before they become active. These modifications will produce changes in the size, the pI or both and can be identified on 2 dimensional gels by the anomalous migration of the protein under investigation.
The technique used currently in this lab for the separation of sample proteins is 2-dimensional electrophoresis (2-DE). This technique uses the power of both isoelectric focusing (IEF) and SDS-PAGE electrophoresis to separate proteins firstly by their pI and then by their molecular weight. For more information on this technique you can refer to our on-line 2DE tutorial.
Once we have our proteins separated we have both a MALDI-TOF and a Q-TOF available for their identification. Digested proteins can be quickly identified by their peptide mass fingerprints (PMF) (see our on-line PMF tutorial) using the MALDI-TOF, or individual peptides can be sequenced using the Q-TOF. If the complete sequence of the organism you are working on is available you can simply use the PMF that you obtain from the MALDI to search the database using a relevent search engine . Even if the organism has not been sequenced you can sometimes get lucky. Below is a gel of Fasciola hepatica excretory /secretory products. This organism has not been part of any sequencing project and yet with what little data is present we were able to identify quite a few of the proteins. The results are being written into a paper at the moment, so we will just give you a taster of what we have found.
Searches using PMFs do sometimes reveal homolous proteins, for instance we have been able to identify calmodulin in Schistosoma mansoni as it has a highly conserved region that is present in a number of organisms. Generally though we have found this not to be the case and so we normally reserve searching with PMFs to organisms that have been fully sequenced. S. mansoni has been partially sequenced so there is some information available and we have found a number of interesting proteins, including a fatty acid binding protein homolog, superoxide dismutase and tegumental antigen Sm20.8 to name a few. But all the same we also had very strong spectra from the MALDI that produced excellent PMFs, which gave no hits, no doubt because the proteins are simply not present in any databases. This is what makes Caenorhabditis elegans such a tempting model, if you have a good, accurate PMF then you will almost certainly get a hit. It is hard not to be enthusiastic about a method that can produce such unequivocable results.
Unfortunately none of the parasitic nematodes that we are interested in as parasitologists have been sequenced fully, which makes protein identification a bit of a lottery. You can always try using the MALDI and you might get a hit if the protein has been entered into the database. This, however, is often not the case and if you are hoping to find homologs, then the hits will have to be very strong ones to be convincing. With the S. mansoni calmodulin the first 5 hits were all calmodulin from other organisms and the first 3 hits had very high MOWSE scores. This is an infrequent occurrence, though and when you cannot get a hit, it is time to turn to the Q-Tof. This mass spec also uses Tof to measure the mass/charge (m/z) of sample fragments, but in this case it also has a quadrupole that is able to separate individual peptides (If you wish to learn more about mass specs then take a look at the ABRF webpage ). After the mixture has passed through the quadrupole the selected peptide can then be fired into an inert gas, which on collision, breaks it up and the m/z of the subsequent peptide fragments measured by the detector. In this way it is possible to obtain accurate sequence information from the peptide (except for leucine and isoleucine that both have the same mass) of interest. Then it is a case of searching through the databases with that sequence to find hits. The sequence that you obtain may not be very long, but you can always sequence several of the fragments to gain extra information. You can also constrain your search so that only proteins of a narrow molecular weight range are searched.
 d |
A Proteomic map of F. hepatica excretory/secretory (ES) products from in vitro culture
|
Tutorial: 2D Electrophoresis for Proteomics
Tutorial: Protein Identification by Peptide Mass Fingerprinting