Thursday, 10 July 2014

Pigment of my imagination again!

The results of our Royal Society-sponsored, Y10 mealworm proteomics experiment are now in hand thanks to the help of Dr. Mark Dickman in the CheLsi group at the University of Sheffield (left). Dr. Alison Nwokeoji in Mark's lab, analysed the samples that Michael and I pooled from the extraction and purification of meal worm larvae using ion exchange chromatography. Just to refresh your collective memories, everyone extracted soluble protein, centrifuged to discard the insoluble debris and handed me their supernatants. I pooled them all and passed them through a 10ml Q Sepharose column during the day. I eluted with a series of increasing concentrations of NaCl, culminating in a 2M NaCl elution for the final step. Having collected the fractions, you then used saturated ammonium sulphate to concentrate the proteins, centrifuged and redissolved ready for SDS PAGE analysis. Since the first gels were a little streaky, we dialysed the samples and the students whose samples we chose to send to Sheffield were: Marcus Kennedy and Millie Keegan from the "Crick Bench" .

The objective of the mealworm experiments has been to establish this simple beetle as a school friendly model organism, with which to illustrate contemporary methods of biochemical analysis. For example, investigations into the mechanisms of cancer have moved towards single cell analysis of the proteome and the genome of individual cancer cells.Of course it is not clear at this stage whether this will prove to be the most incisive or fruitful approach to the attack on cancer (or even an understanding of the fundamentals of the disease), but it is the methodology that is current and therefore I feel for this reason alone,  its inclusion in our innovation lab programme is justified. 

In the mealworm proteome project, we set out to develop a simple, reproducible

protocol for the extraction of soluble proteins from the mealworm (larvae at first). The prominent bands isolated from Marcus and Millie's dialysis samples (prepared from the whole class extraction and purification) were analysed by Mass spectrometry in the Sheffield. The whole process represents an example of how proteins are analysed in complex mixtures and simulates contemporary analysis of for example protein biomarkers in blood or other biological fluids.

So, how successful were the students? If we break the workflow down into stages:
1. Mealworm "husbandry" 
2. Protein extraction
3. Protein purification
4. Protein concentration/de-salting
6. Mass spectrometry
7. Data Analysis and interpretation

Then the students collectively score 8/10 (well maybe 7/10 since data interpretation is still in hand). The only step that wasn't completed by a Y10 (just for those who don't know, Y10 students are typically 14-15 years old and have had very little laboratory experience), was the mass spec analysis, since this is a very specialised process. However, the analysis of proteins by mass spectrometry is becoming more common place and is often outsourced by professional laboratories to experts like Dr. Dickman.

What did we discover? The main protein that I want to mention represent the tip
of the iceberg that is the mealworm genome and proteome! The protein identified from the low salt elution fractions of Molecular Weight in excess of 150 000, turns out to be a "melanization" related factor from Tenebrio. It has been seen in related organisms, but how robust is the association of function to this predicted protein sequence? 
It is one thing to say a protein sequence is like another one, whose function is known, but it would be premature to assign function without further research and experiment. Why? Well, assigning a function for a molecule that is identical to a known protein of the same sequence is a good first step in assigning function, but does the protein have the appropriate properties? Well, it did have the characteristics of a phenol oxidase (it appeared to be oxygen sensitive and showed a brown colouration). The molecular weight on the SDS Gel was close to the mass of the best "hit" by MS and previous studies have shown this activity to be present in the larvae.  One pure fraction, remains unedentified until we obtain a complete genome sequence for Tenebrio (and I am working on funding for this), but we will follow up analysis of this by other methods. The other proteins that were identifies are also linked to melanization and so I have an interesting weekend ahead on PubMed! 

These data will form the basis of the first of two publications to emerge from the innovation labs at the UTC. The Y12 work on the non-ribosomal peptide synthetases has led to the amplification of the gene from Synechocystis and we are currently aiming to produce the recombinant protein products in a number of wild type and reconfigured forms for the development of novel specificity enzymes. The summer is the time for working through the data and when we have completed this, I shall let you know the details of our findings. In the meantime, thanks to our collaborators (Mark and Alison), our sponsor, the Royal Society and the enthusiasm of the Y10s. I would also like to single out Jack Webster a member of the Greenland Biodesign team in Y12, who has made all of the mealworm research possible through his dedication to developing and maintaining our UTC colony. 

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