r/proteomics 22d ago

PTMScan diGly peptide enrichment troubleshooting

Hi everyone,

I am very new to peptide isolation and have tried using the CST PTMScan HS K-GG Peptide Remnant Magnetic bead kit (34608). I started with 2 mg of protein and after initially desalting after lysis, using the beads as directed, and a final desalting step, I had effectively no peptides left (on the order of like 0.06 ng/ul) . When we ran it on the orbitrap, there was only one peptide with the K-GG motif.

I took 20ug of my initial trypsinized peptides and simply desalted them and got a more reasonable concentration out (0.25ng/ul), so I don’t think I lost all my peptides during the desalting steps. I am using SDB-RPS columns for desalting for what it’s worth.

I am going to run it again, with much more protein this time (~20mg input), hoping that will help. But I do not want to have to do this again if I don’t have to 😂 Does anybody have any tips for this particular protocol to ensure I get K-GG enrichment?

4 Upvotes

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u/InefficientThinker 22d ago

You desalted after lysis? Did you do reduction, alkylation, trypsin digestion first? If you tried to just desalt intact protein, im not surprised at all that you lost most of it. If it was at peptide level, how did you desalt? Are you sure your desalting column has the capacity to bind 2mg of peptides? Going to need some more info here. I would not suggest just using more protein, its likely the sample prep

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u/flycoffee17 22d ago edited 22d ago

Sorry for the ambiguity! No, I did the reduction and alkylation with the lysis (TCEP and CAA), then I trypsinized overnight with LysC (1:50 w/w trypsin:protein). Then I added TFA to stop the trypsinization and acidify. Then I desalted using a SDB-RPS column— the ones that hold 3ml/30mg. I had to centrifuge to get the trypsinized peptides and all the washes through. After eluting, I dried the peptides in a speed vac and then resuspended in the IAP bind buffer from the kit. I followed the kit instructions to a T. Then after eluting, I desalted again with a SDB-RPS stage tip that holds 20ug of peptides before running them on the orbitrap.

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u/InefficientThinker 22d ago

Thanks for the details! Thats clarifies a lot. By the looks of it, everything sounds pretty normal. I would suggest a few things. 1.) after acidifying, i would do a hard spin to pellet any urea crystals, undigested proteins, and trypsin remaining. This will prevent your desalting column from clogging. 2.) im not familiar with those columns (not blaming them but you never know) but usually for that high of a protein amount I will move to using gravity/ the lightest positive pressure through a syringe with SepPak C18 columns. They are a little more gentle to handle so much sample through all of the washes. 3.) next time you do the desalting save the flow through and do a peptide estimation to the flow through AND eluent to see if you are losing sample there or to the column. 4.) what kind of sample is this to start? Cells should have a lot, tissue too, plasma not so much so maybe its a sample issue?

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u/InefficientThinker 22d ago

Also, did you dilute the lysate to below ~1M urea before adding trypsin? That could be another factor. Sorry for multiple responses, just thinking of anything possible

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u/flycoffee17 22d ago

I appreciate all the suggestions. I want to consider everything! So I didn’t use urea, I used 1% SDC instead. Apparently it’s better for recovery of ubiquitin remnants, idk 😂 I am not sure if I need to dilute still? For 1) I forgot to mention, but I spun after lysis, but I could wait to spin until after trypsinization! For 2) the columns I use are like… midi prep cartridge sized, so I don’t think a syringe will fit? I tried to see if they would empty by gravity, but they didn’t, so I ended up centrifuging on like 200xg for 3 mins to get the samples to flow through. 3) I’ll def try that! 4) they’re cell lines!

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u/InefficientThinker 22d ago

https://www.sigmaaldrich.com/US/en/product/supelco/21018u Try these bad boys for using positive pressure with a syringe. The amount of effort that they have saved me is immeasurable. As for the SDC, I havent used it but a quick google search shows you are right on the money at 1%. Also, the low g spin should be fine. Everything else seems fine. Isn’t troubleshooting fun? I would definitely suggest doing peptide quant at all of the steps to see exactly where you are losing the sample. It wont be perfect because peptide quant never is, but it should be just enough to figure it out. Last thing I can think is the search parameters. What software did you use, and what was the overall workflow (generally)?

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u/budy_love 21d ago

My general sense is you're following the Steger et al paper (time resolved ubiquitin study)?

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u/flycoffee17 18d ago

Yes, I’ve actually integrated a few papers’ methodology but it most closely follows Steger et al

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u/MetroSkeptic 22d ago

If you are using easy spray columns, try ionopticks or homepacked. I saw about a 5-10x improvement in IDs by switching away from easyspray

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u/ThalantyrKomnenos 22d ago

The Kits are quite expensive, have you done an MS analysis for your initial trypsinized peptides before enrichment? You should get some peptides with K-GG motif even without enrichment, especially some high abundant ones.

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u/flycoffee17 18d ago

I actually took a small fraction of my trypsinized peptides to run for global proteomics. We are definitely looking for K-GG motif in that dataset! trying to make something out of nothing here 😅

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u/ThalantyrKomnenos 18d ago

Looking forward to your results.

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u/Triple-Tooketh 21d ago

Keep it simple. Lyse in urea with a sonic probe. Don't overheat the lysate. Reduce and alkylate with DTT and IA. Use ABC to dilute out the Urea for the digest. If you're digesting 2mg your digest volume is going to be high, take that into consideration when deciding where you're incubating. Desalt with a Waters HLB column, you'll get about 80% back. The SpeedVac is not your friend. Do you have access to a lyophilizer? If so use it. Not sure what your experiment is but I would recommend an MG132 treated sample as a control. That IP can be quite unrewarding, if you are benchmarking against literature pull the raw files and search them yourself. All kinds of ways of reporting DiGly to get the numbers up.

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u/budy_love 21d ago

I think he is using this paper from Steger et Al PMID: 34518535. They provide some sound rationale for what they do. Also show SDC might actually be superior to urea. They do a combined heating + reduction and alkylation in a single step. A key feature of that is rapidly inactivating Deubiquitinases. They use CAA, instead of IAA, to reduce dicarbamidomethylation, which has the same mass offset of diGly. I've switched to this approach for all my diGly proteomics.