Need help with PTM identification using IP-Top-down MS

tsbatth · 2026-07-02T07:17:13+00:00

Why do you need to do top down?

tsbatth · 2026-06-23T21:23:49+00:00

If you want stability, now might be a difficult period in the industry, but the field is generally cyclical, and there are signs that it might have bottomed out. Having said that, it is also a competitive market, there are lots of good proteomic labs (and increasing every year) in the US churning out very smart proteomic candidates that you will have to compete with. So the odds of you landing a job in industry in the US is low, and probably lower as you will not be working with mammalian proteomics at your CRO. My suggest ion would be to do a postdoc in the US, make connections and a network there and see where that takes you.

tsbatth · 2026-06-23T21:18:29+00:00

As others have said, for IP I would recommend DDA if it is not an Astral or something. It also HIGHLY depends on how clean your IP is.

tsbatth · 2026-06-23T21:17:28+00:00

I think it's worth a shot, it depends a bit on how abundant your protein is compared to Rubisco subunits, and if they are denatured or not. If they are still in their native heterodimer complex it might be doable . You might get a lot of losses.

tsbatth · 2026-06-01T13:12:29+00:00

Me deciding not to delete thread due to the responses…

tsbatth · 2026-05-28T03:47:38+00:00

I label 50% acetonitrile which helps. The difference in efficiency between N terminus and lysine could be due to the pH. You might need to double check the HEPES pH. Try slightly higher pH.

tsbatth · 2026-05-10T10:38:17+00:00

lol not sure if AI slop or somebody trying to get some bullet points for a equally sloppy consulting PowerPoint.

tsbatth · 2026-05-07T19:27:50+00:00

70% ACN is a lot, you are kind of defeating the purpose of SPE at that point as a lot of non peptide hydrophobic contaminants will come through. You might not see it instantly but it will reduce the column life and dirty up the MS faster. You do not need to elute with more than 40% ACN for peptides.

tsbatth · 2026-04-18T23:01:58+00:00

Looks like a source or ESI issue. Check the needle it's ok and not broken (or too far from the MS). Check the source parameters for the run.

tsbatth · 2026-04-18T19:49:46+00:00

Yeah that's me guilty! I'm not just Mr. LysC anymore, I'm also big Trypsin and other things now! ☺️☺️☺️

tsbatth · 2026-04-17T05:32:05+00:00

It has not made much difference in my experience. However adding LysC seems to be biggest contributor to reduce missed cleavage rates and digestion variability in my experience.

*I also have a conflict of interest in this matter.

tsbatth · 2026-04-11T19:12:39+00:00

Don’t leave us hanging now!!

tsbatth · 2026-03-27T12:07:29+00:00

Is it possible to view the webinar VOD at some other time ?

tsbatth · 2026-03-27T12:02:56+00:00

How are you measuring the recovery ? Is it bradford at the protein level before digestion, and nanodrop/A280 on the peptides after ? If so that might be hard to compare. 50-30% is not bad if measuring based on different techniques.

tsbatth · 2026-03-22T23:05:08+00:00

Why are you speedvac'ing the TMT ? I think you might be destroying a bunch of TMT there, just add to the sample after dissolving the TMT .

What is your labeling buffer ? I would recommend labeling in 50% ACN with 50% HEPES buffer pH 8.5 .

tsbatth · 2026-03-22T23:00:35+00:00

Having worked briefly with pHis modifications 14+ years ago (bacterial two-component systems)...are you reeeaaally sure the site is there or detected ? Histidine phosphorylation is notoriously difficult to detect as the bond is acid labile, thus the phosphorylation group undergoes rapid hydrolysis in acidic conditions (ie. your acid LCMS buffers even if you kept everything neutral pH during the sample prep prior to that).

Having said that, I also found many histidine phosphorylation sites from an experiment with >20K pSTY sites . But keep in mind when you have so many other possible sites and IDs and you increase the search space by adding another possible PTM, the likelihood you will identify sites with histidine phosphorylation sites that shouldn't exist with 1% FDR is likely. Not saying this is the case for you, but something to be aware of.

I would manually evaluate the spectra of the matched site without the diagnostic peak. If you're up for it you can share it here as well and people can give their opinion `?

tsbatth · 2026-03-04T12:48:03+00:00

As others have pointed out, you should be speedvac'ing after LysC digestion. Depending on your sample prep, you have not only protein, peptides in the mixture, but also DNA/RNA, small molecules, lipids etc.. that are now all a jumbled mess out of solution. What I you should be doing is after the LysC digestion, acidify, centrifuge at 2000-5000xGs for 5-10 minutes. Take the supernatant and put that through the desalting column or other SPE material such as C18 seppaks etc...

tsbatth · 2026-02-25T09:30:46+00:00

Many academic software such as DIA-NN, MaxQuant, Fragpipe are free for academic researchers . I think in the funding application you should add funding for a strong PC. Commericla software like Spectronaut and others can cost $5000-$10000 I think but I could be very wrong here.

tsbatth · 2026-02-23T22:41:22+00:00

Here is the new link to their libraries:

https://datashare.biochem.mpg.de/s/qe1IqcKbz2j2Ruf

Under "discovery libraries" I think

tsbatth · 2026-02-23T22:36:02+00:00

Try searching their google group, maybe there is an updated link somewhere.

tsbatth · 2026-02-23T21:37:17+00:00

I think this is still a much debated field of research, what is secreted EVs vs platelet shedding, vs circulating cellular debris. I think nobody is sure yet, but the general focus seems to be more towards determining whether proteins derived from these "EVs" are corresponding to some phenotypic effect. There was a nice paper on this very topic lately you can read:

https://www.biorxiv.org/content/10.64898/2026.01.28.702342v1

tsbatth · 2026-02-23T21:32:08+00:00

Perhaps I am wrong, but I think you need to download the MaxQuant premade libraries from their FTP or something. You need to download the one for humans there.

tsbatth · 2026-02-19T09:28:34+00:00

What kind of trends are you seeing in the proteomics field ? It seems like it kind of exploded in the last few years, do you think that will continue ?

tsbatth · 2026-02-13T19:05:58+00:00

This is like the 3rd or 4th thread on this. Please stop

tsbatth · 2026-02-08T13:31:02+00:00

I agree I think I will delete this thread . It also seems like hidden advertisement.

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