Hi Noah,
Since you are having good results w/ HeLa, but not the fat tissue I would
suspect sample prep could use some tweaking to accommodate the fat tissue ...
though I have no idea what your method is. You could compare your procedure
with the EasyPhos method here: Humphrey et al., “High-Throughput and
High-Sensitivity Phosphoproteomics with the EasyPhos Platform.”
(https://doi.org/10.1038/s41596-018-0014-9). It may be that using SDC, and
SDB-RPS SPE clean up method could improve your results. I've used the EasyPhos
protocol a couple of times on cultured cells with good success, but not on
tissue. However, I use SDB-RPS extensively for SPE because it eliminates
neutral and negatively charged/hydrophobic materials e.g. detergents and
phospholipids very well. If you don't like making stage tips, you can swap out
the stage tips for Waters Oasis MCX products into the workflow.
Best regards,
Brian
Rest of post
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4
<email obscured>>
Sent: Thursday, December 10, 2020 1:13 PM
To: <email obscured> <email obscured>>
Subject: [ABRF Discussion Forum] What are people's favorite phosphopeptide
enrichment methods and protocols?
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Hi,
Despite having worked in this area for a long time, I'm struggling with a few
finer points of phosphopeptide enrichment.
1. I thought our TiO2 and newer CaTiO3 methods were pretty fool-proof, but I
have some new people struggling to get high reproducibility.
Do people have experience with some of the pre-packaged, optimized systems,
like the Pierce High Select Fe-NTA columns?
How about magnetic bead systems vs spin-filters vs batch? Does anyone have a
clear preference either for ease of use or robustness and efficiency?
2. We have been working with some samples derived from fat tissue and are
getting horrible phosphopeptide enrichment. A positive control with equal
amount of HeLa peptides looks fine.
Anyone have any thoughts why this should be? I would have thought that any
lipids, including phospholipids would be lost to the C18 during pre-enrichment
clean up.
Thanks,
Noah
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