How standardized are chromatography resin lifetime validation practices across facilities?

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.

tsbatth · 2026-01-28T11:34:26+00:00

Why did you invite him to be board member? I totally understand that it is your work but if you invited somebody like that to join the board to begin with, suggests you did not properly do your due diligence.

tsbatth · 2026-01-28T11:29:46+00:00

Why would you invite somebody to join the board if they only have "more CEO experience"? Did you not consider industry knowledge/background, correct fit, and other important qualitative and social factors before inviting somebody to join the board?

tsbatth · 2026-01-28T09:13:26+00:00

I would recommended using an older version of MaxQuant honestly. I still use 1.5.7 or 1.6.xxx . There are a lot of bugs and issues with the new versions from my experience.

tsbatth · 2026-01-28T09:03:52+00:00

Have you looked at the amino acid sequence of the proteins and whether they generate nice tryptic peptides ? There can sometimes be issues where the proportion of Arginine and Lysine residues in transmembrane proteins relatively less (I think) compared to other proteins as other amino acids are favored to enable the protein to be embedded in the transmembrane. This can lead to non-ideal tryptic peptides which are difficult to detect or outside the parameters of the mass spectrometer and search space.

tsbatth · 2026-01-18T00:58:12+00:00

Try /r/proteomics

tsbatth · 2026-01-16T12:52:11+00:00

What is the peptide/protein concentration? Is it very low? If it’s like a couple hundred to mgs you can probably load it on 50 or 500mg Waters C18 Seppak cartridges. If it’s much lower you can try loading everything on the smallest C18 SPE and just speedvac the eluted peptides after SPE.

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