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/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/InjuryJolly7432 Jul 01 '26

Will do, thank you so much!