Can you provide more details about your sample preparation?

It is a standard protocol we have used successfully in the lab before where we fish for binders using a probe, digest the proteins on the beads with trypsin O/N and desalt/concentrate the samples using C18 stagetips. Attached also the chromatogram of one of the samples. My guess is that somehow the MS/MS scans cannot be assigned to a peptide?

Hi,

That sounds strange indeed.

A few questions:

- Did you manually check some of the MS/MS spectra to look at the quality of those?

- What search parameters did you use? For example, what mass accuracy, etc. Does the accuracy you're searching with match that of the (calibrated?) instrument, both for your precursor and fragment ions?

- Did you include the relevant fixed/variable modifications in your search, like cysteine alkylation (if that was part of your sample preparation?

If you've performed a similar experiment before, try and look up a few peptides that were used to identify a protein in your previous, successful, experiment and see if these are observed in your second run as well, to compare if anything is different between the runs.

Hope this helps!

Thank you for your answer Eef Dirksen

There was a problem with precursor selection for MS/MS which we are looking into now.

Hi Nila,

How about the collision gas? Did you have enough pressure to fragment the selected ions?

Hediye.

Hi Hediye, an electric board of the mass spectrometer was broken so at least we know what the problem was! Thank you.