Dearests, I am working on Proteomics and want to check the immune response (changes) brought by a challenge from a pathogen to the honeybee larvae based on hemolymph samples.
Furthermore, I want to do the study in the LC/MS system.
I have also collected all the required hemolymph samples.
How is it possible?
Can you please suggest me on sample preparation for this study?
Really thank you for your advice and valuable help.
Hi, I know nothing about bee immunity, but I can suggest proteomic methods. First, you can do comparative proteomic approach - you compare proteomes of control and infected hemolymph. Alternatively you can try quantitative proteomics (which allow you to obtain relative quantification of proteins between control and infected hemolymph) but this approach requires more sophisticated methodology (like iTRAQ). If you are just interested in overall differences of hemolymp protein composition, take equal amount of it, lyse the cells, purify the protein (using precipitation) and run a SDS-PAGE. Silver stain the proteins, digest them in the gel with trypsin, extract and analyze with LC-MS/MS, searching your data against bee database. If your MS instrument is sensitive you should be able to obtain data on proteins associated with immune response. Please refer to general guidelines on gel-based proteomic aproach.
Dear Wojciech Cypryk, I would like to appreciate for the advice you suggested and I found it valuable. But in our lab, we do have the latest LC-MS/MS technology and very good sample preparation protocol which is reputable and we are not using SDS-Page based analysis. The problem is I don't know how to compare the availability of immune responsible genes/ associated proteins in the samples. If it is like comparing total protein against the differences among immune associated proteins in both groups, It is fine. But, if there is some other techniques that I can use proteomics to carry out immune response experiments, please dearests, give me your valuable advices.
Thanks for your understanding and support
I agree with the answer of Wojciech Cypryk. In case that you want to detect relative changes in proteins related to immune response against any type of external agent, I would suggest you to perform an iITRAQ labeling (4-plex or 8-plex). After labeling you will be able to observe all relative changes in the proteins caused by the challenge. Then you need to see from all those proteins which are related to the immune response.
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