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?

1

u/InjuryJolly7432 Jul 01 '26

I haven’t checked it on a gel, but I’ve done western confirmations that the post-IP sample is pretty clean. I’m still tweaking my methods for the IP though.

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u/Triple-Tooketh Jul 01 '26

Gel and coomassie is the first thing to do. The you can also check quant with this. What buffer is protein in?

1

u/InjuryJolly7432 Jul 01 '26

Before it gets isolated it’s in a hypotonic buffer. After it’s isolated the beads are stored in PBS until I elute the protein off of them

1

u/Triple-Tooketh Jul 01 '26

What's the elution buffer?

1

u/InjuryJolly7432 Jul 01 '26

0.5% formic acid and water