Hi  Neha

i would recommend to use the swissprot database for your search, you might have more success.However, I think at the end it is not the database but rather the sample preparation, it is very difficult to match proteins from 1D Gels using MALDI-TOFs, the complexity is still far to high. Could you provide some more details regarding your experimental setup, this could help to provide more detailed help.

best regards

Bogdan

Don´t know if this is helpfull, but when you are using Mascot you can also specify a taxonomy (for example Viridiplantae) this will exclude a lot of not necessary genomes. However, if your microalgae has not a fully sequenced genome it will be very difficult to get some hits with mascot. Maybe you could check for the genome on Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) or you may check one oneKP (https://sites.google.com/a/ualberta.ca/onekp/system/app/pages/search?scope=search-site&q=microalgae) whether there is trsnscriptome data for your organsism, however you will need to translate it into protein models. Additionally I totally agree with Bogdan, it will remain difficult anyway and won´t be only a matter of the database. Do you have the possibility to switch from Maldi to ESI MS? This would at least provide better raw data. Dont´t feel shy to get into contact for further questions. Good luck!

Sebastian

Hello Bogdan,

Thank you for your answer. I'll try and match my proteins with SWISSPROT database. The peaks of the sample are very clear and sharp. I have eluted differential proteins from SDS-PAGE and digested then with trypsin. In the publications related to proteomics of algae, most of then have matched their proteins to NCBI database or chlorophyta database.

Thank you Sebastian for your descriptive reply. The microalgae I am working on is a novel isolate - Scenedesmus sp. so it doesn't have sequenced genome. I got some matches of my proteins but they are not of algal genome, could I still use them as reference? or the proteins should be specifically of algal genome.

Unfortunately, I do not have an ESI-MS facility available at my Institute.

Hi Neha,

I´m afraid this won´t give you a reliable result. Matches for not-related genomes are either highly ambiguous or represent only a few "lucky" shots against evolutionary very strongly conserved protein domains. For the study of differential protein expression this is not really a recommended data situation. So I suggest to check for transcriptome data to try to build a protein model database out of it. Further you could additionally try to use Peaks Studio Software (https://www.thermofisher.com/order/catalog/product/PEAKS40) and do de novo sequencing with your data, meaning you don´t need a protein model database to interpret mass spectra . However, this is a very complex task and you may ask someone for support, unfortunately I´m not familiar myself with that software.

Good luck!

Sebastian

Hi Neha

If, your peaks are intensive enought, I would recommend to try to sequence them and then blast for homology. After that you could try to confirm by an antibody based system

Thank you Sebastain for your suggestions. Even I was thinking of performing De-novo sequencing.