How to use ISOGlyP?
The input sequences can be sent either through the text box area or uploaded as a plain text file. Either way, the sequences must be in FASTA format. For glycosylated S and T, you must replace S and T by $ and +, respectively. After the input sequence(s) have been entered, the next steps are to select the prediction constraints, the ppGalNAc transferases, and the number of columns in the output. Finally, input sequences have to be submitted for processing, and the program will display the results on a new webpage. These steps are described below. For more information about the basis of the ISOGlyP program, please refer to the Background page
- Sequence Input - The ISOGlyP Graphic User Interface (GUI) provides two input options: uploading a file or directly entering sequences in FASTA format.
Option 1: Uploading a file - This option allows the user to upload sequence(s) saved in a text document for prediction. Every single sequence saved in the text document must be in the FASTA format for processing.
Option 2: Copy/Paste sequences - With this option, the user will either directly type or copy and paste the amino acids into the box for processing.
For both options, the maximum number of input sequence(s) that can be processed by ISOGlyp depends on available memory on the server. The current ISOGlyP server can handle up to 200 input sequences in a single request. This number may vary (mostly increase) depending upon the amount of available memory. There is no limit on the length of the input sequence(s).
- Prediction Constraints - The user can choose prediction constraints by checking the boxes below the XXXXX-T/S-XXXXX sequence. Although enhancement values are available for +/- 5 residues we believe that utilizing a +/-3 X residue window will reduce 'noise' in the calculation. This is because to date transferases have been relatively insensitive to the flanking 4-5 N- or C- terminal X residues, i.e, have enhancement values ~1.
- Cys/Thr Values - Cys and Thr value enhancement settings are made below the C/T values pull downs. As there are no determined enhancement values for Cys or Thr, their default values are chosen to be 1.0. However, since these residues are homologous to Ser structurally there is an option to set their values to those of Ser.
- Ser Weight - By changing this weighting parameter, it allows for global changes of the enhancement value product (EVP)for each Serine site. Values calucluated from the enhancement value tables will be multiplied by the inserted weighting factor. (Any negative values will be ignored.)
- Select Isoform - The ppGalNAc T isoform enhancement values to use are chosen by checking the boxes below the ppGalNAc Transferases heading. To date we have data for ppGalNAc T1, T2, T3, T5, T10, T11, T12, T13, T14, and T16. The MAX selection returns the max enhancement value product (EVP) from the selected ppGalNAc T isoforms. As data for additional ppGalNAc T isoforms are obtained their values will be added to the site.
- Output - The number of columns in the output are adjusted by the most right box. When the no more than 3 isoforms are selected, the result for each isoform is displayed in one column. Otherwise, 3 columns in the output file should be optimal.
- Submit - The sequences can then be submitted for processing by clicking on the submit button, which will transfer you to a new window displaying the prediction output. The output page gives the residue number, flanking sequence, and the enhancement value product (EVP) based on the user entered constraints. The higher the EVP value, the greater likelihood of that position being glycosylated. Note that the results can be exported in an Excel spreadsheet format.