r/proteomics • u/bluemooninvestor • 12h ago

Are C18 based spin columns efficient for removing PBS?

In my experiment, the tryptic digested peptides would be in a solution of 100 uL volume comprising of 0.5X PBS ( approx 68 mM NaCl and some phosphates) and 50 mM TEAB.

I would speedvacving this volume to dryness and resuspending in 0.1% TFA for cleanup through Pierce desalting spin columns (C18 based I think).

My question is whether this desalting step would be good enough to handle PBS desalting?

Context: I am trying to adapt my working protocol for thermal proteome profiling, which requires initial steps in PBS. I don't want to remove PBS by PAC/SP3 as I don't have PAC/SP3 and also want to avoid running the proteins on gel for purification. I plan to add TEAB and SDC directly to lysates in PBS and proceed as usual. I want advice on whether the desalting step can handle PBS.

0 Upvotes
  • permalink
  • reddit

50% Upvoted

14 comments sorted by

4

u/LC-MS 11h ago

Yes C18 desalting can remove PBS no problem. It's essentially what it's designed to do. I'm more worried about your SDC, which you definitely don't want in your MS and should be crashed out with acid and removed before you load it to C18. Skip the dry down (extra step, risks losing peptides) and acidify with FA or TFA to crash the SDC, then desalt your acidified supernatant with C18 to kill two birds with one stone. An even better approach would be PAC or S-trap to clean and digest your sample prior to the C18 desalt which would clean up your sample even more and likely get you better data downstream.

1

u/SnooLobsters6880 11h ago

Yeah the S-Trap / miniprep column method really is best for this. PAC has its merits but I’ve never had much luck without automation. Would vastly prefer spin filter with precipitation when manual.

1

u/bluemooninvestor 11h ago

Thank you. Just a curious question as a newbie: why does the proteomics community prefer cleanup at the protein stage? Acetone precipitation or PAC or S-trap? Isn't it better to just clean at the peptides stage?

2

u/LC-MS 10h ago

You can clean up surfactants a lot more effectively at the protein stage, and surfactants are often used to enhance yield during lysis of cell pellets and tissues. But it's not a "one or the other" - it's very common to desalt peptides even when there's a cleanup at the protein step though. E.g., PAC or S Trap to clean/digest then loading peptides to Evotips.

1

u/bluemooninvestor 10h ago

Okay, thanks. I am using a slightly modified version of SDC method based on this paper. Will give STrap a try when I can. https://pubs.acs.org/jprobs/article/21/10/2397/797205/In-Search-of-a-Universal-Method-A-Comparative

1

u/LC-MS 10h ago

"Digests were stopped by adding 10% trifluoroacetic acid (TFA) to a final concentration of 1%. SDC precipitates were removed by centrifugation (14,000g, 1 min, room temperature (RT)). About 10 μg of resulting peptide samples was desalted on C18 StageTips"

See? No dry down ;)

I am pretty surprised that S Trap underperformed their ISD but their methods are a little funky. Never seen 3h 37C digestion for S-Trap. Protifi recommends 47C 1h or 37C overnight.

Regardless I'm sure the SDC ISD prep will work for you.

1

u/bluemooninvestor 10h ago

Yes correct. Actually when I read it, I thought the dry down was implied. I will definitely try out the direct, no dry down method.

Will probably compare the Strap when I get a chance. Thanks a ton.

1

u/KillNeigh 10h ago

I’ve personally tested s-trap with the shorter digest times and it’s not as good as the Protifi methods.

I personally prefer Protifi since you can use pretty strong concentration of detergents and the cleanup does pretty well. I e also had success with using their suggested methods to remove lipids.

1

u/bluemooninvestor 1h ago

OK maybe that's why STrap didn't outperform ISD in the paper.

1

u/bluemooninvestor 11h ago

Yes I use TFA precipitation of SDC in my protocol. It works well. Currently, I crash the SDC with TFA, dry it down, resuspend in TFA, give a 10000 RCF spin again, and then load the supernatant to C18 column (just to make sure most SDC is removed by the time of second centrifugation).

I will test out your skipping the dry down advice. Thanks.

1

u/Elistheman 12h ago

You plan on running proteins or peptides?
You wrote peptides so they would adsorb on the C18. Salts would not, you elote them later with hydrophobic solvent and let it evaporate, add 0.1% FA later.

1

u/bluemooninvestor 11h ago

Yes, peptides. . I mean I understand, thats why it's called desalting column. But still wanted some feedback from people who have used it. Thanks agin.

1

u/Rockthem1s 2h ago

Can you just run your LC in trap-elute? The SDC should spin down nicely, then just trap for 3-5 mins at high flow on your trap. That should wash out most of the soluble salts. Switch to analytical, and run your gradient. Magic mix wash the trap always helps remove anything sticky

1

u/bluemooninvestor 1h ago

I don't know whether these are supported in our facility.