Hello,
I have exosomes isolated from serum of patient cases and would like to use them for proteomics analysis. Previously I had used RIPA lysis buffer to lyse my exosome specimens and that worked well for western blots. However, our core facility for mass spec informed that RIPA would not work for MS analysis and would need a detergent removal prior to injecting them for MS. Is there any other buffer that would work well for exosome MS or is there any other process that could be substituted for detergent addition to lyse the exosomes? Any suggestions would be very helpful.
Thanks,
I'm going to make two assumptions here:
1. You have an exosome pellet after using ultracentrifugation to isolate them from serum.
2. The MS facility there can digest and extract peptides from SDS gels.
if these are true, you can use SDS -PAGE to prepare a sample, and sumit the gel piece to your core. Follow this method.
Add ~100 ul of 1%SDS/20mM Tris, pH 8-8.5 to the exosome pellet, after removing as much liquid as possible. Vortex well, then heat at 85C for 10 min to lyse the exosomes and denature the proteins. Do a BCA protein microplate assay at 1:4 dilution. Calculate the volume needed for 5 ug of each sample. Dilute with Laemmli buffer with bME, DTT or TCEP, heat again to reduce disulphide bonds, and load on a mini gel (i.e. AnyKD TGX gel, BioRad). Only run sample about 1-1.5 cm into the gel. Stop the electrophoresis, fix, stain, cut out the middle 1/3 lengthwise of each short lane, put in a tube, and submit to the MS facility.
This approach ensures all the protein is solubilized, and denatured for efficient digest from the gel matrix. The SDS is eliminated during the fixation and staining of the gel, and also during the washing and digest of the gel pieces.
If you want to get more protein IDs, and cost is not too much, you can run a longer gel with more protein (~15 ug), and cut out multiple segments across each lane. Generally, the more fractionation, the more proteins that are detected by MS.
You can skip the protein assay if you are normalizing by volume of serum or some other factor. But i would for sure run and stain a full test gel on a practice prep to be sure you don't have mostly serum proteins in the prep, which we see a lot in plasma/serum exosome samples submitted for MS in our Proteomics Core.
What protocol do you or your MS facility plan to use for protein digestion for MS? In-gel, in-solution digest or FASP? Depending on the method you can select from several widely used lysis buffers like 8 M urea/2M thiourea-based or 4% SDS buffer (highly recommend with FASP).
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