3.8 Database
Generation
for Targeted Protein
Search
1. Create an empty file in Excel or an alternative spreadsheet
editor. Place taxonomy identifiers (taxid) from the roll-up
results in the first column and the DBType code specifying
the UniProt database type in the next column, do not include
headers, and save as a tab-delimited text file named targeted_database.txt. DBType options are defined in the ProteoClade
user guide; the “a” option is used to download all available
proteins entries in a fasta format.
2. To download the protein databases from UniProt, execute the
following command:
download_uniprot_batch(“targeted_database.txt”)
The individual fasta files will be stored in the fasta’s directory with the taxid number as file name.
3. Combine all individual species databases into one file:
merge_fastas("targeted_database.fasta")
At this stage, additional fasta files can be included when
placed in the fasta’s directory.
3.9 Targeted Peptide
Identification
1. Configure search engine for the use of the created sequence
database. For this step, the use of MaxQuant is suggested,
which is a freeware, mass spectrometry platform-independent,
and in addition to peptide identification capable of various
methods of protein quantification.
2. Installation and user instructions can be found at http://www.
coxdocs.org/doku.php?id¼maxquant:start
3. Guidelines for MaxQuant settings for large database search: use
a smaller database for a first round of search; limit the size of a
database to a maximum of 400,000 protein sequences (~100
organisms); set missed cleavages to zero or one; limit the
number of modifications, minimum peptide length of 7–8
AA; and disable the MD5 option under advanced global parameters to save processing time.
4 Notes
1. UA and UB solutions must be freshly prepared and used within
a day. Do not heat urea-containing buffers to avoid the introduction of unwanted amino acid modifications.
2. IAA solution must be freshly prepared and used within a day.
Alternatively, use 0.5 M stock solutions in water stored at
À20
C.
3. C18-StageTips with three layers of Empore material (3 M) can
easily be assembled according to the protocol developed by
Rappsilber et al. [13]. Alternatively, ready-to-use C18 tips are
commercially available from various manufacturers (e.g., Supeltips, ZipTips, or Spin-Tips).
176
Sjoerd van der Post and Liisa Arike
Generation
for Targeted Protein
Search
1. Create an empty file in Excel or an alternative spreadsheet
editor. Place taxonomy identifiers (taxid) from the roll-up
results in the first column and the DBType code specifying
the UniProt database type in the next column, do not include
headers, and save as a tab-delimited text file named targeted_database.txt. DBType options are defined in the ProteoClade
user guide; the “a” option is used to download all available
proteins entries in a fasta format.
2. To download the protein databases from UniProt, execute the
following command:
download_uniprot_batch(“targeted_database.txt”)
The individual fasta files will be stored in the fasta’s directory with the taxid number as file name.
3. Combine all individual species databases into one file:
merge_fastas("targeted_database.fasta")
At this stage, additional fasta files can be included when
placed in the fasta’s directory.
3.9 Targeted Peptide
Identification
1. Configure search engine for the use of the created sequence
database. For this step, the use of MaxQuant is suggested,
which is a freeware, mass spectrometry platform-independent,
and in addition to peptide identification capable of various
methods of protein quantification.
2. Installation and user instructions can be found at http://www.
coxdocs.org/doku.php?id¼maxquant:start
3. Guidelines for MaxQuant settings for large database search: use
a smaller database for a first round of search; limit the size of a
database to a maximum of 400,000 protein sequences (~100
organisms); set missed cleavages to zero or one; limit the
number of modifications, minimum peptide length of 7–8
AA; and disable the MD5 option under advanced global parameters to save processing time.
4 Notes
1. UA and UB solutions must be freshly prepared and used within
a day. Do not heat urea-containing buffers to avoid the introduction of unwanted amino acid modifications.
2. IAA solution must be freshly prepared and used within a day.
Alternatively, use 0.5 M stock solutions in water stored at
À20
C.
3. C18-StageTips with three layers of Empore material (3 M) can
easily be assembled according to the protocol developed by
Rappsilber et al. [13]. Alternatively, ready-to-use C18 tips are
commercially available from various manufacturers (e.g., Supeltips, ZipTips, or Spin-Tips).
176
Sjoerd van der Post and Liisa Arike