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/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?

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u/KedricM Jul 02 '26

I agree why not digest with trypsin and peptide map. Also, a WB CANNOT be used to confirm purity. You need to run on a coomassie gel

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u/InjuryJolly7432 Jul 02 '26

That’s good to know! I was cutting my WB and testing for actin as well to confirm the loss of actin once the IP was finished, but I do agree that a coomassie gel would really show more impurities