We are an EV-based lab and characterize the EVs using MS to look at the protein content of the EVs.
My question is why do these biological replicates have such a different elution profile? It seems like the sample at the top completely lacks hydrophilic peptides compared to the bottom one (the first half of the chromatogram).
Is this the MS sample prep error or the heterogeneity of EVs? - I don't think either as for the MS sample prep same stock solutions were used and regarding the heterogeneity of EVs - these are from wild-type untreated mice and therefore, cannot show such stark differences in their profile. However, I might be wrong and further insights are highly appreciated.
Details on the sample and MS sample prep method used (Note: Both the samples were prepared at the same time using the same stock solutions)
Any leads are highly appreciated.
Hi Chintan Bhavsar,
have you tested the reproducibility of your EV isolation procedure by WB with i.e. CD63? From the first view it seems like you have a variation in the sample prep method. I would suggest to first check the reproducibility of all method steps (EV-Isolation, protein extraction from EVs, digestion and desalting of peptides before comparing samples. Usually the LC-MS itself is not producing that much variation.
Best,
Murat
Yes, we have our EV isolation protocol well standardized with western, size distribution well characterized. We also have checked the reproducibility for our MS protocols and that is also standardized as well.
These are the reasons why we are surprised with the results.
This is strange!
From the first view it looks as if different amounts of proteins or even different types of samples are injected in this both samples.
Have you already analysed the raw data?
You may spike digestion control protein (for sp3 workflow) along with a spike in the hydrophilic peptide (for QE HF) both to verify the sample prep and/or MS bias...
İsmail Emir Akyildiz that is really a good suggestion. I would try this out
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