Hello friends,
During proteomic analysis of stem cell secretome, we observed that secretome is significantly enriched for PFAM protein domain (kinesin motor domain). I was also wondering about presence of some intracellular signalling and receptor proteins (may be by shedding) in secretome. When I searched literature then I came across a thesis and one paper published in reputed journal where people have also reported findings like this. What can be the possible explanation for the presence of these proteins in the stem cell secretome.
Are these proteins getting into secretome in secretory form?
Are stem cells shedding receptors?
or packaged vesicles in secetome are transferring these proteins from cell to cell?
Please provide your valuable insights.
Thanks!
Hi Ajay,
Thanks for sharing your results on this topic. I'd like to have a little more of information on how you performed the proteomics analysis. Regarding that, try to think that the secreted vesicles were vesicles inside the cell. And these vesicles' positioning and transport is probably mediated by kinesins! So, depending on how you did the proteomic, you may still see some kinesins associated to the early secretory vesicles and see them in your pull-down or mass spec analysis. I wouldn't jump to the conclusion that these vesicles contain kinesins inside, unless strong evidence is provided. Also, most of the kinesins are made of coil-coiled structures, which are really sticky, so you may want to rule out the possibility of contamination (doing an experiment on the importance of those kinesins in the secretory pathway of your vesicles? over-expression and see co-localization?). I hope this could give you some insights on what I know about the field of kinesins ;)
Cheers,
Charly
Hello Charly,
Thanks for your beautiful answer and it did clear my confusion upto some extent about presence of kinesin in secretome.
I am almost certain that there was no any contamination. Lemme explain the protocol to you. I did incubation of stem cells at 70% confluence for 48 hrs. After end point I collected the secretome and centrifuged for 10minutes at 5000 rpm. That rules out the presence of any cellular debris in secretome. Also remanant cells in flask were checked for any cell death and apoptosis and were found to be healthy. Then I concentrated around 140ml secretome to 4ml and then did protein extraction from that secretome. The extracted protein was resuspended in PBS and sent for LC-MS/MS analysis which was performed on WALTERS platform using uniport database.
I hope this will make you clear that there was no contamination. Please have a look on these links where people have also observed kinesin in secretome but a proper explaination for the same is lacking
http://recipp.ipp.pt/bitstream/10400.22/4737/1/DM_JoanaCosta_2013.pdf
https://www.ncbi.nlm.nih.gov/pubmed/21860587
After looking at the links, you will be clear that it's not a contamination or by chance thing. I was also wondering on the presence of receptor and intracellular signalling proteins. It will be great if you can provide your valuable inputs regarding same.
Thanks & Regards,
Ajay
Have you considered, that exosomes in your culture supernatent might be the source of the proteins that surprised you ? exosomes you will only get rid of by ultracentriguation so 5000 rpm will not get rid you of excosomes. , cheers Michael
Hay Michael,
I was actually not interested to get rid of exosomes. I wanted to delineate the whole secretory profile.
Cheers!
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