Let's try something new as well. Anyone interested, please follow me on Bluesky:
@proteomicsmarburg.bsky.social

@pastelbio You have some invite codes @neely ? :)

@translational_proteomics @UCDProteomics You can do it in 1x PBS or 4% SDS/HEPES buffer, I prefer second.

@pwilmart @neely @UCDProteomics don't forget the pie charts and some 5 directional, scaled Venn diagrams.

@pwilmart @UCDProteomics

@kabalak @UCDProteomics 😅 it was just about the CAM abbreviation! I was quite sure, you're talking about some special depletion method I don't know 😏 what do you dilute the plasma with?

@kabalak @UCDProteomics Ok, I need help here. Probably a problem with abbreviations, but I have no idea what you mean by CAM process. :D I searched the whole literature in order not to look like a moron, but I didn't find anything. The only thing that comes to my mind is Carbamidomethylation, but then, I don't understand how you do it directly on plasma.

@UCDProteomics @pwilmart you can then spend 400k years, trying to deconvolute the signal from a particular single cell in your sample.

@translational_proteomics @UCDProteomics Prepping plasma? I thought it is always ready to use ;-) Normally, we just dilute strongly (1:10 for human) and start the CAM process, followed by SP3 cleanup .

@pastelbio a link please?

@kabalak @UCDProteomics
How do you prep the plasma before starting with SP3? 👀

@kabalak @UCDProteomics i always have way higher losses with StageTips so I prefer peptide SP3. I also tried direct MS, and if you wash much more extensively on protein level, it's fine. Here they describe a direct MS https://www.nature.com/articles/s41596-018-0082-x but there are many more examples.

@UCDProteomics will do tomorrow! It's 22:00 here now 😅

@UCDProteomics In my hands, the additional precipitation of peptides on sp3 beads didn't really cause any significant losses of peptide IDs, but created a cleaner sample, so I can protect the column better.

@UCDProteomics so... in the very original paper (that didn't work), they did the first round of sp3 on the lysate, then digestion on beads, and then, recapturing of the peptides on the beads (what I call peptide sp3). Many papers afterwards skip the peptide sp3. They just take the supernatant after the digestion and either do c18 or just straightforward SpeedVac the sample and resuspend in FA or TFA for MassSpec.

@UCDProteomics do you do the sp3 on protein level only or do you proceed with peptide as well or you skip the peptide sp3 at all?

Found an old spectrum from 2011! :D

@pwilmart remember the original sp3 protocol that doesn't work? 😏

Most failed proteomics methods are things that sounded good on paper but something was fundamentally wrong or the technology was not capable. I have no idea how random enrichment of protein noise from plasma ever got off the ground. It sounds ridiculous on paper. Let's see if some nanoparticles with different surfaces can have a really rare peptide stick before the crap from albumin saturates everything. Maybe there are non-random patterns in the random binding. Time to go commercial…

New paper published with the involvement of our lab!

https://pubmed.ncbi.nlm.nih.gov/37607954/