Brett PhinneyThis is from the SP4 paper https://pubs.acs.org/doi/10.1021/acs.analchem.1c04200 , which is really good btw . When ever I see optimization steps like this, it just screams to me that this is very hard to reproduce across labs/people. I generally think it's a good trade to trade sensitivity for reproducibility in most cases which I why I like using the King Fisher and Magnetic beads over doing it by hand
Witold Szymański@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?
@translational_proteomics not sure I understand . What is sp3 on the peptide level ? I don’t think I’ve heard of this 🤔
@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 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.
@translational_proteomics oh very cool ! I didn’t know that ! Can you share your protocol ?
@UCDProteomics will do tomorrow! It's 22:00 here now 😅
@translational_proteomics haha thanks !!
Fatih Demir@UCDProteomics @translational_proteomics Directly StageTip'ing will be the most favored way, though I like doing the SP4 (aka SP3 peptide cleanup) just to avoid the SpeedVac limbo. But are people really doing a digest and completely drying in the SpeedVac & then going for LC/MS? Brave people, thou shall be for that. #proteomics #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.
@translational_proteomics @UCDProteomics In my personal experience, the yield from SP3 peptide cleanups is generally (much) higher than for StageTipsy. Interestingly, this discrepancy seems to be dependent upon the original sample source matrix (plasma vs. tissue vs. pull-downs...).
@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 .
@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.
@translational_proteomics @UCDProteomics Erhm, simply diluted plasma, treat with TCEP/CAA, boil at 95 °C and you have your CAM=carbamidomethylation. Why should you not do it straight?
@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?
@translational_proteomics @UCDProteomics You can do it in 1x PBS or 4% SDS/HEPES buffer, I prefer second.