Hello,
I submitted few samples (2 cell lines, 2 serum exosomes (healthy), 2 patient serum exosomes) for mass spectrometry, in order to see if we could see relevant exosome proteins and associated biomarkers. My cell lines picked up a good amount of exosome proteins, along with CD81 and CD82 [Exosome markers], but my serum exosomes (healthy and patient) did not show any exosome proteins, instead picked up a lot of serum proteins with very high amounts of albumin and immunoglobulins. Is there any way to get rid of albumin/IgG from exosome preparations?
Does performing an albumin/IgG depletion step work on serum exosomes if I want to identify low abundance exosome proteins on mass spec? If so, is there any specific kit that works best?
Any answers or suggestions would be hugely helpful.
Thanks,
Hi Sneha,
yeah, depending on your isolation procedure, there might be tons of albumin and other highly abundant proteins in your serum EV preparations.
Running albumin/IgG depletion should principally work, but we’ve never tried it because it’s pretty expensive.
Another (and less costly) approach would be to just use an isolation method that produces less contaminated vesicles or to further purify the vesicles you already isolated.
This might mean isolating EVs using a “clean” method such as SEC, density gradient centrifugation or immunoaffinity (if that’s an option). Alternatively, you could use an additional purification step (probably also SEC or density gradient) for your previously isolated EVs (I suspect these are from precipitation or differential UC?).
Good luck,
Dominik
Thank you very much for your answer Mr. Dominik. Yes, I did use ultracentrifugation + ultrafiltration for exosome isolation from serum and cell lines. I was unable to process this sample using density gradient, since the amount I was starting with was just 800 ul of patient sample, and sucrose gradient would result in a high loss of exosomes, and I would not be having enough for downstream processes. And, we do not have a core facility that offers SEC for me to perform it.
Also, I was not sure if the albumin/IgG depletion step would work because I was skeptical if exosomes are attached with albumins or immunoglobulins in some way or the other, or if this was just because serum by nature contains abundant amount of albumins and immunoglobulins.
Dear Sneha,
Hi. I suggest to read the paper of Zarovni, Corrado et al. Methods (2015), 87:46-58. From there, and from many others articles it appear that inmunocapture can be the solution to your problem with ultracentrifugation, by making in addition isolation in a one step. There are several commercial its at this aim, which are able to minimize albumin and inmunoglobulin contamination. Best regards.
Daniel
Hi,
May I ask what is the volume of your starting material for both cell lines and serum? I am isolating exosomes from plasma using SEC but I also have no exosomal markers by mass spec and alot of immunoglobulins and albumin.
Hello, Ms. Sara,
My starting volume for cell lines are 100 ml and for plasma I use 800 ul. I see CD81 marker on my cell lines, but my serum/plasma does not indicate any exosome markers.
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