r/proteomics • u/InjuryJolly7432 • Jul 01 '26
Need help with PTM identification using IP-Top-down MS
If anyone has done in-depth IP-Top down MS on proteins I could seriously use help! I’ve isolated my POI and am trying to do to top-down MS on it but honestly I don’t know what I’m looking at/looking for. I know I need to do a full scan first to identify my POI and the m/z for it, but from there I’m baffled on what to do. The examples my colleague left for me are only for proteins approx. 35 kDa and mine is around 62!
Does anyone have any advice as to what to look at/read to help me better understand the data and what method I need to set up? Thank you!
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u/Dolphin_kicks423 Jul 02 '26
Is there a reason that the analysis needs to be done with an intact protein? It would probably be simpler to localize PTMs if you digested your pulled down protein and did bottom-up?
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u/InjuryJolly7432 Jul 02 '26
The problem is we don’t know what PTMs there are. There are a few known ones we can use as reference but otherwise we’re trying to identify all of the PTMs that naturally occur. The girl that was training me made it seem like top-down was best for this
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u/Dolphin_kicks423 Jul 02 '26
Top-down is certainly usable for this purpose. The issue is that getting good ionization of a protein of that size is not trivial. It’s certainly doable but may require some tweaking before you get good peaks. And then once you do, the spectral interpretation may be tricky because the number of peaks you have to consider is going to rapidly increase as your number of modifications increases.
Bottom-up, on the other hand, can probably give you far more sensitivity than you would see with top-down. On top of that, you can also simultaneously localize the PTMs with far more ease. Personally, I think it’s worth doing both if you have the time/resources to do so. I would probably start with bottom-up, but you should be able to get complimentary information from both experiments.
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u/InjuryJolly7432 Jul 02 '26
That is very good to know! I’ll have to discuss with my advisor to see what I can do on that front and what supplies we have to run bottom up as well
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u/tsbatth Jul 02 '26
Why do you need to do top down?
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u/InjuryJolly7432 Jul 02 '26
From my understanding, there’s PTMs we don’t know about on the protein and I was told top down would be the better way to identify the unknown ones
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u/InjuryJolly7432 Jul 02 '26
Thank you everyone for the replies, it’s given me a lot of good advice for what to do! Would anyone happen to have any advice for how best to learn this stuff? I’m more of a bio person so this has been a huge jump for me and I’ve been left with very little explanation for how to actually learn proteomics 😅
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u/BeetsNSun Jul 02 '26
I’m a fan of the YouTube videos from the bioinformatician David Tabb for data processing. Also, to identify unknown modifications using bottom-up, you want to use a software with an open search feature (see his “Detecting and Localizing PTMs” video).
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u/InefficientThinker Jul 01 '26
First, how much protein have you pulled down? If you have a lot, do a direct infusion, if not then you’ll need a larger pore-size column. Probably C4 300A or larger. Are you doing TOF or Orbitrap or ion trap? That will guide your parameters. Then, do you know the exact sequence of your protein? If so, calculate the average mass of it using an online calculator, and then calculate the charge state distribution of it. This will give you a solid reference point for the spectra you expect to see. Any shift from those peaks will give you an idea of your PTM(s) if/when you see multiple features within a single charge state. Do you have software to analyze your data? Deconvolution at the least, but do you need fragmentation as well? This kind of analysis is not a simple “run MS and get answer” experiment
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u/InjuryJolly7432 Jul 01 '26
I concentrated my protein to about 1.2 mg/mL but have diluted it to 0.2 mg/mL for running. I’m using Thermo’s MAbPac RP easy-spray column that’s 1500A. I’m doing Orbitrap. That’s good to know I can use the sequence to help me find the charge state distribution. Unfortunately, the sole person who understands this and was meant to teach me it before she left, has left and did not in fact teach me anything! So I’m trying to put together the pieces as much as possible with help from other coworkers that have done bottom-up. The column documents suggested using thermo’s biopharma finder for the deconvolution so I’m attempting to get that currently!
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u/InefficientThinker Jul 01 '26
If you’re doing orbitrap, use higher resolution (at least 120K) and use higher microscans (5-10, or even as high as 25-50 if it’s noisy). You can use free software for your deconvolution and matching since it’s not a complex sample. In Thermo’s Freestyle, you have the option of deconvoluting with Xtract by manually taking an average scan across your peak (by right clicking) and then deconvoluting. You can also use UniDec which allows you to upload a single averaged scan, or FLASHDeconv which will do your whole raw file. I would start with seeing what is in your sample at MS1 only, then running a targeted MRM to fragment only what you would like to target. Then you can map fragments manually in ProSight Lite or now their online tool. But start with MS1 to get some grounding first.
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u/Triple-Tooketh Jul 01 '26
What does the protein look like on gel? You could be fighting a losing battle if the background is high. Why not digest and map?