Any chance of doing tandem MS of your samples instead of PMF? It works only well if the proteins in the respective bands are pretty pure. How good or bad look the spectra? Which instrument and database search engine did you use?

What were the mowse scores for the identified proteins?

I used the Applied Biosystems Voyager-DE STR and NCBInr database. The spectra looked good after I tried different intensities to make the baseline flatter and peaks higher.

MOWSE scores are 93, 114, 102, 110, respectively. Others are lower than 80.

Which proteolytic enzyme did you use for protein digestion? Did you use correct parameters for database search? Did you try any other database?

Trypsin was used for protein digestion. I used the default parameters firstly and changed the parameters when I did not get the expected result.

The scores you have stated above are significant although but it can't be guaranteed that the identified proteins are the correct one's. In my work, I got wrong protein hits when I used wrong parameters in the database search. You need to take into account the protein modifications and the no. of expected missed cleavages. I think MS/MS won't be of much help here but you may wish to give it a try. Overall, the situation is bit weird. I don't have experience with bacterial proteins, instead I used to digest and analyze model proteins, e.g. Cyt C, Caseins, B-lactoglobulin etc.

Thanks. Actually I found the weird result may come from the wrong protein complex that I purified.

Might be a dumb question, but are you sure your proteins of interest are in the database you used? Is this bacterium already sequenced or did you search for similar organisms? The scores between 90 and 120 are o.k., this is what I would expect with PMF only data. Where was your threshold for p=0.05? Around 55? This would mean that even the ones with scores around 80 would be significant hits, not great, but likely. But in case actual protein is not in the database you might pick up something completely random which is just close to what you are looking for.

Another point you migth want to check is the following: If you prepared the gels by yourself, set carbamidomethylation as variable modification and add propionamide-modification as additional variable modification on cystein. Self-casted PAA gels are full of monomeric acrylamide which readily reacts with free thiols generating mixed modifications on the cystein. Typically you can spot this in the data as double peaks for Cys-containing peptides with an un-annotated one 14 Da higher than the annotated one.

>Thank you Marcus. My bacterium has been sequenced before. As for threshold for >p=0.05, I cannot find it in the Matrix Science website. Other insignificant results were >about 50~60 scores.

>I made the 15% SDS-PAGE gel by myself. Will the acrylamide remain in this gel of >high concentration? And I washed the gel in pure water for 1h before doing mass spec.

Dear Didi,

p=0.05 is usually the green shaded area on the left hand side of the graphics for the score distribution. If you did not change anything, this will be the default. The significance threshold relates to this value and is usually in the range of 50-60 for mammalian samples. The size of the search space influences this value, the smaller the search space, the smaller the threshold value, thus when restricting the search on a specific bacteria it will be lower.

The acrylamide will remain in the gel, washing before the digestion will not help. The modification takes place during the gel electrophoresis (once you reduced the proteins in the loading buffer and applied them to the gel in basic buffer, the alkylation will start). This can be prevented by doing the alkylation before applying the samples to the gel, yet one have to be careful as using e.g. IAA releases hydroiodic acid (HI), which will change the pH of the loading buffer causing trouble in the electrophoresis. Typically, ready made gels are less critical in that regard as those are extensively washed and do not contain a lot of monomeric acrylamide anymore.

Regards, Marcus