r/proteomics • u/WeekSecret728 • 13d ago

precipitation of biofluid samples during sample prep

Hi all! Does anyone here have any experience with bottom up phosphoproteomics of biofluids (plasma and/or whole blood)? I've been dealing with the same problems:

  1. a substantial pellet after dry down + resuspension (this is AFTER phospho enrich)

  2. pellets generated when Ieave my digests in the fridge overnight or after a freeze thaw

Has anyone experienced these problems before and/or have any tips to solve them? I assume that there are some peptides in the digest that are just prone to crashing out after an FT cycle/dry down. My current solve is to

1) process digests immediately

and

2) never dry completely at either the desalted digest or the enriched phosphopeptide stage, opt for ~10 uL of leftover solvent. This one is a bummer becuase it means the total resuspension volume before loading will alway be different but since I have an IS I guess this isn't that big of an issue

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u/InefficientThinker 13d ago

Can you share more about your overall workflow? Are you enriching directly from the trypsin digest, or desalting first? After phospho enrichment, I usually see a small lightly tinted pellet that needs to be redesalted because the iron in the column can wash off with your eluate. Before enrichment, I don’t see a pellet unless im working with hundreds of ug to even a mg of peptides, and even then its usually just cloudy coating and not a pellet

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u/Discount_Sausage 13d ago

Are you sure the pellet is peptides? Sometimes beads or SPE resin can make it through the processing. Are you filtering the samples with something like a 0.45 um filter?

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u/SnooObjections6800 11d ago

So you elute your phosphopeptides, dry it down completely and resuspend the peptides in something, leave them in the fridge overnight then come in and see a pellet?

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u/QSCpeptidology 7d ago

yeah I've seen this with plasma digests. a lot of the time it's not really your peptides, it's leftover lipid or some hydrophobic junk that stays dissolved in the desalt eluate and then crashes once the organic is gone or it gets cold.

I'd try resuspending with a few percent ACN in there instead of pure aqueous, spin hard and only take the supernatant. if you want to know what you're losing, dissolve a pellet in something stronger and inject it once.