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Tutorial:
To begin with, a protein sequence file in FASTA format is needed. For example, the RANGAP1 FASTA file can be downloaded here.
1. Specify the type of modification for calculation.
There are three different kinds of Modification types available: Sumoylation, Ubiquitination or no modification. Using the modification type drop down menu, choose one of the modifications to proceed. When either sumoylation or ubiquitination is chosen, the output only contains peptides that contain a potential sumoylation/ubiquitination lysine residue in such peptide. When no modification is selected, m/z values of all peptides in the input protein are reported.
2. Input the protein sequence(s) of interest.
There are two possible ways to input a protein sequence into Ubl Finder:
(a) A protein sequence in FASTA format can be input into the text window.
(b) A protein sequence file in FASTA format can be uploaded using the browse button.
3. Specify search parameters.
For each of these parameters, the goal is to limit the number of m/z values being output. Since there is often times a limit for the number of m/z values that can be input into the inclusion list of a mass spectrometer, these parameters would help to output a range of m/z that have a better chance of identifying the peptide(s) of interest.
There are 5 search parameters:
Maximum Modified Residues: This parameter specifies how many modifications are possible for a particular peptide in the input protein sequence. By default, this setting is 1 since the chance of two sumoylation/ubiquitination sites showing up in one peptide is very low.
Maximum no. of Miscleavages: This parameter specifies how many miscleavages are allowed. By default, this setting is 1.
Display Ions: This parameter specifies the charges of the peptides to be reported in the result page. By default, +2 and +3 are chosen. This means that for each of the peptides in the protein, two m/z values are reported.
Specify m/z Exclusion Limits: This parameter specifies the range of the m/z values to be reported in the result page. By default, the full range is reported.
Separator: Either tab or comma delimited output can be chosen.
4. Review all search parameters are correct and hit search button.
Result:
For each of the proteins, the title of the protein from the FASTA file is reported.
To use the result in a mass spectrometer, simply cut and paste the values under "all m/z" into the inclusion list of the spectrometer.
A list of peptides with their corresponding m/z values are also reported. For sumoylation, if a consensus sumoylation site (hydrophobic KX(D/E)) can be found, the residue number is reported.
A coverage map is also included for easy visualization of the coverage of the output m/z list where " * " denotes that particular residue is included in the m/z output list and " - " denotes the residue is NOT included. For the lysine residue(s) that is not included in the output list " ! " is used to denote such a lysine.
Fraction coverage specifes the percentage of the lysine residues being covered in the m/z list for a particular protein. A fraction coverage of 1.0 represents a 100% coverage.
Parameters:
Choose from a drop down menu for the available modification. Currently, Sumoylation, Ubiquitination and no modification are available. When either sumoylation or ubiquitination is chosen, the output only contains peptides that contain a potential sumoylation/ubiquitination lysine residue. When no modification is chosen, m/z values of all peptides in the input protein are reported.
Protein sequence(s) of interest is needed for calculation of the m/z values. This program only allows FASTA format. Multiple sequences can be input at the same time.
Upload Protein Sequence FASTA file:
Instead of pasting a sequence into the text box, a FASTA file can also be uploaded using the browse button.
This parameter specifies how many possible modifications can be found in one peptide.
For example:
AGCTYKAKAK
* * For this peptide, there are two potential lysine residues for modification, denoted using *. For max. Modified Residues of one, only one m/z value (for each charge chosen) is reported. If two are input, two m/z values will be reported for this particular peptide.
This parameter specifies how many possible numbers of miscleavages are allowed.
This parameter specifies the charges of the peptides to be reported in the result page.
This parameter specifies the range of the m/z values to be reported in the result page.
This parameter specifies the output format as either comma delimited or tab delimited.