AMPK Methods and Protocols

3 Methods

3.1 Collecting
the Pathway
Components

Identify the pathway of interest and download the involved protein

sequences in FASTA format. End with a nonredundant list of

proteins for downstream analysis (seeNote 2).

1. Visit the KEGG pathway web site athttp://www.genome.jp/
   kegg/pathway.html. SpecifyHomo sapiensas the species of
   interest by setting the “select prefix” tohsa, and enter the
   term “AMPK” into the keyword field. Run the search and
   select the AMPK pathway (hsa04152) from the result list
   (Fig.2). This will lead you to the web page describing the
   human AMPK pathway in greater detail.


2. From the link list in the top right corner of the AMPK pathway
   web page, follow the link “KEGG GENES.” This will result in
   a list of genes associated with this pathway (Fig.3a). For the
   human AMPK pathway, the list contains 121 proteins of which
   each is briefly described.


3. To retrieve further information about individual genes of inter-
   est, follow the corresponding link. You will be guided to a new
   web page, at the bottom of which you will find the amino acid
   sequence of the human protein together with the coding
   sequence. Click on the tab “AA seq” and save the protein
   sequence in FASTA format (Fig.3b).


4. A bulk download of all genes is a bit more complex, because
   KEGG currently provides no option for this.Steps 5– 11 can
   be used as a quick work-around.


5. Open a text editor and copy the list of all genes and their
   description into a text file.


6. To access the individual fields in the gene list, you can use the
   bash functioncut. Alternatively, import the text file into a
   spreadsheet, e.g., Microsoft Excel. Choose “Delimited” as
   field separator and specify the delimiter as “space.” This will
   place the KEGG gene identifier into the first column of your
   spreadsheet and protein KO identifiers in second column.


7. Copy the contents of the first column, open the UniProt [15]
   cross-referenced databases (http://www.uniprot.org/data
   base/), and click on “Retrieve/ID mapping” in the menu bar.


8. Paste the KEGG identifier list in the search field. Select KEGG
   as database from where you have obtained the sequence identi-
   fier and UniProtKB as the database to which you want to
   convert your identifier. Upon hitting the “Go” button, a
   results page opens providing the cross-references (Fig.3c).


9. Activate the “Filter by Reviewed” to reduce the output to the
   120 Swiss-Prot entries (seeNote 3). This step will get rid of

Tracing AMPK Evolution 117