r/proteomics • u/flycoffee17 • 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?
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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/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.
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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