How to use SEQUEST to search a nucleotide database

One nice feature of the SEQUEST MS/MS cross-correlation program is that it can translate a nucleotide sequence to a protein sequence, allowing you to search raw nucleotide databases, like the many available from the NCBI website.

Allrighty then, so how do you use SEQUEST to search a nucleotide database?

Well, that depends. If you are using the SEQUEST Browser, on the run SEQUEST page, you’ll see a radio button allowing you to select the type of database to search (Auto, protein, nucleotide). It’s right under the database dropdown box.

If you are doing your search in Qual Browser Xcalibur 1.2 there is also a radio button for you to select the type of database in the SEQUEST or TurboSEQUEST parameters dialogue box.

However, if you are trying to do the search from the Qual Browser in Xcalibur 1.1 there is no button to select protein or nucleotide databases. The default setting is protein. Therefore you must edit a text file called sequest.params. The sequest.params file is found in the /Xcalibur/results directory on your hard drive (e.g., C:/Xcalibur/results). Edit the file with a notepad. You’ll see a line like this

nucleotide_reading_frame = 0 ; 0=protein db, 1-6, 7 = forward three, 8-reverse three, 9=all six

When nucleotide_reading_frame = 0 a protein database is assumed. For nucleotide searches, set nucleotide_reading_frame = 9, unless you absolutely know the reading frame of your system under study. When you’re done, save the text file as sequest.params. If you want to switch back and forth between protein and nucleotide you’ll have to keep editing the sequest.params file. Incidently, if you use the SEQUEST Browser or SEQUEST within Qual Browser Xcalibur 1.2 then clicking on the nucleotide button will set nucleotide_reading_frame = 9.