Hi
My ‘MS-compatible Silver-stained’ protein bands produced high quality MALDI-TOF/TOF spectra (many decent peaks with strong signals at both the MS and MSMS level), but are unable to match any proteins from its own Species/Genus.
The entire sample processing and mass-spec workflow should be alright as other samples (from popular species) produced significant matches with high scores.
At the Species level, my samples are from a bacteria with a complete proteome in Uniprot; whilst up to the Genus level, there are at least millions of protein entries.
I have tried various other approaches, but there is no improvement in the match:
(1) Mascot searches against protein and EST databases, respectively;
(2) Mascot ERROR tolerant search;
(3) De novo peptide sequencing then MS-BLAST
Should I go for a Mascot search against its genome? or What could be the possible reason behind?
Thanks.
Were the proteins from whole cell lysate or else? how do you sort out real signals from background (even though they are clear signals).
What about the mass error tolerances for the search? Are they too strict and the measured masses are falling outside that range due to instrument drift?
In line with Matthew's point: does each run contains an internal standard for calibration? This can reduce the error margin.
Hi Matthew and Ke-Wei,
Before each job run, the MS and the MS/MS were calibrated using the peptide calibration standards.
Our other samples (from human or mouse, as well as b-gal peptide standard) spotted around those bacterial samples all show matches with high score.
Their error margin seems reasonable (the instrument drift should be low). The workflow and settings thus seems alright.
Hi Kay,
If you are sure about the quality of MS and MS/MS spectra, then you can try a search using the online version of NCBInr database, dont restrict the taxonomy ... Search it using "All entries".
Also you can try searching once with - "Semitrypsin" for enzyme specificity...
I hope this helps..
All the best
Saravanan K
Have you looked at the MS/MS spectra to see if there is a good ion series? Can you see y1 at 147 or 175 if this is a tryptic digest? Some peptides ionise awesome in MS only to fragment like crap. The 842Da autocatalytic trypsin fragment is a good example.
HI,
A couple of questions
- what MS and MS/MS tolerances do you use?
- what is the MALDI instrument?
- you performed both peptide and protein analysis, right?
There is one thing which I would like to understand: you tried the procedure (from gel to Maldi plate) with other proteins too. Do I understand correctly?
Because if you run a standard next to your sample on the gel and the standard does not give any trouble... Then the most simple conclusion is that something is happening in one of the steps before.
In case you did NOT run a standard on the gel, then I would start with a very simple test. Take a known protein (e.g. BSA) and run your procedure once again.
At the same time, prepare the same protein solution without doing any gel run.
Compare the two dataset. If they give similar results, then you can conclude that your initial dataset is fine. Otherwise, you should try to figure out what kind of large protein modification happened at some point in your sample prep.
It would be goo if you could share a concise summary of your sample prep. Maybe we can figure out what happened.
F.
Dear Fabrizio,
The MS and MSMS tol uses are 75ppm and 0.2Da, respectively. We used the ABSciex 4800 MALDI TOF/TOF.
Appreciate your suggestions. We have standards of known proteins that are processed along with our samples (from gel plugs).
The workflow should be alright (for mass-spec), while there might be something happening at the sample preparation, gel running, and staining.
I discuss with my colleague, whom conducted the upstream gel handling part.
Thank you.
The MS tolerance is OK but the MSMS tolerance is too small. The fragment ions are not in a well compressed packet when leaving the collision cell. I would expand this tolerance out to at least 0.6 Da. 0.2 Da is OK for a Q-TOF but not a TOF/TOF.
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