Peptides may vanish due to losses during affinity cleanup or incomplete elution from C18 columns, and trypsin inactivation at high digestion temperatures. Optimize digestion conditions, improve peptide recovery, and use spike-in controls to identify and fix the issue.

What do you mean by "We checked the digest on a gel, it is complete"?

Are the proteins intact or digested?

Do you add some acid after digestion to inactivate trypsin and precipitate undigested proteins?

I meant that the protein is fully digested, there are no bands. We stopped the digest with formic acid.

Which acquisiton mode do you use?

Considering you are having a plasma background, do you detect any plasma peptides in the end?

You could try spiking a standard protein like BSA in the beginning to test that the workflow works in principle:

(1) If you do not detect a good amount of BSA peptides, something in your sample preparation is likely wrong.

(2) If you do detect a good amount of BSA peptides, the problem probably has to do with your antibody (detection).

I use basically the method I set um together with a thermo employee in a training session: Acquire X Deep Scan with dynamic exclusion, charge state 2-5, Intensity threshold 8.0 e3, MS1 resol. 120,000, MS2 resol. 15,000, HCD 30%.

I saw plasma peptides from this kind of samples in the beginning, but not anymore. The "background signal" of plasma proteins returns when I digest the 5 fold amount of sample. It seems that I lose much more material during sample preparation than in the beginning.

Considering the protein concentration of our samples, the end volume and the injection volume, there should be about 0.8 µg of peptides on the column if there were no substantial losses.

Annemarie Lippert, there are a few possible sources of Your problem.

1) You mentioned problem with the sensitivity loss of the instrument. Are You sure, that this was resolved? Can You compare a standard sample (e.g. BSA digest, standard mix, etc.) from before the service and after to see if there is any signal intensity loss?

2) Something (was) changed in the method. I would start with checking ion source settings, as, at least on Thermo instruments, You can quite easily go from having them set in the method to using ones set in acquisition window. Just double check all method parametrs in the process. BTW, do You use exactly the same method for standard mix and sample analysis?

3) I would really avoid running samples with even "only a small amount of CaCl2". Not good for You mass spec. I had situations where even trace addition of salts impaired method sensitivity (even for a few runs, then effect could be gone due to washing out the salt with consecutive runs) I would also rather not use BCA for peptide concentration, as there are colorimetric quan tests for peptides available.

4) This one is not so obvious, but I see that You are using AcquireX Deep Scan, which is kind of DDA method. I've never used it, but from what I understand it automatically creates some inclusion and exclusion lists. Maybe Your peptides were somehow filtered out (as a background)? I would try to go with "normal" DDA acquisition, or even better, set up a targeted method for expected masses. Just make sure Your MS is well calibrated.

It is never easy to remotely pinpoint the problem with MS method (so many factors), but I hope that this answer will give You some kind of a clue. ;)

Good luck!

Thank you very much for your suggestions.

Yes, I have a targeted method that I wish to fine tune and use in the end, but it does not give me much data as long as I lose so much of my samples. Right now it helps to see some matrix peptides.

We got a slight improvement by applying less harsh denaturation conditions and are now tackling the concentrations and proportion of DTT and IAA.

By the way, does anyone of you know why there are protocols with and without quenching the alkylation with DTT? DTT interferes with peptide assays, so it is not desirable to have it in the digested sample, but many protocols add it to inactivate IAA, presumably to protect the trypsine from alkylation. How do you handle that?

Annemarie Lippert, we also had issues with too high DTT and IAA concentrations, leading to inefficient digestion and low peptide identifications. We were using 62.5 mM DTT and 128 mM IAA and now use 12 mM DTT (@ 37 °C for 30 min) and 40 mM IAA (@ 20°C for 45 min). Also adding sodium deoxycholate (5% (w/v)) before DTT and IAA treatment can further improve digestion and peptide detection. Sodium deoxycholate is an ionic surfactant that slightly denatures and solubilizes the proteins which facilitates the reduction of disulfide bridges and alkylation of cysteines, as well as tryptic digestion. You need to dilute the sodium deoxycholate to 1% (w/v) before tryptic digestion and you can remove it after digestion by precipitation at acidic pH. For more details you can refer to the methods section of https://doi.org/10.1101/2024.11.25.625156

Annemarie Lippert, to address Your question about DTT quenching: it is probably an artifact from older protocols, which were generally not as fast and used large excess of IAA, thus the risk of overalkylation was serious.

BTW, have You tried using S-trap for sample prep? I've been using it for some time now for global proteomics and it greatly improved my coverage (comparing to FASP), particularly regarding highly hydrophobic peptides.

Thank you both! We are currently performing experiments to improve reduction and alkylation conditions. Hopefully, this will result in better recovery.

I did not come across S-trap before, but we also do not use FASP. Our protocoll is completely in solution, excluding the desalting step with C18 filters in the end.