※ User Guide:

Frequently Asked Questions:

1. Q: Is GPS 6.0 much better than the old version?

A: Yes!
(1) GPS 6.0 has better predictive performance. First, we trained a general model based on 3 types of machine learning algorithms (penalized logistic regression, light gradient boosting machine and deep neural network) using 490,762 non-redundant p-sites. Then, we used transfer learning strategy to construct the kinase-specific models for predicting kinase-specific phosphorylation sites, which improved the predictive accuracy and robustness.
(2) GPS 6.0 deleted some kinases with low confidence of its kinase activity.
(3) The web server of GPS 6.0 can provide more visualizations and is more user-friendly. Users can select different modules to meet different conditions, for example, the id-search module could be selected to perform a task according to the input protein name(s).

2. Q: How to use GPS 6.0 webserver?

A: First, you could find the prediction website in "WEB SERVER" page of GPS 6.0. Second, enter protein sequence(s) in fasta format, which starts with a '>' followed with protein/peptide name. Then, select the kinase node(s). The predictor contains 10 S/T groups, 1 Y group and 2 Dual groups for dual-specificity kinases. Just click the "Submit" button, wait a moment, you can get the prediction results of kinase-specific phosphorylation sites on substrate.


3. Q: How to choose the different prediction modules?

A: We provide 5 modules of prediction for users. You can click "here >>" at "WEB SERVER" page to change the online service mode or just click the following names:
(1) GPS Web Server (LightGBM): The default web has fast speed and visualization function. We provide 3D structure, statistics and disorder propensity of protein.
(2) GPS Web Server (Integrated): The deep-learning prediction balances the accuracy with speed.
(3) GPS Web Server of Comprehensive Prediction: The comprehensive prediction has annotations of secondary structure and surface accessibility.
(4) GPS Web Server of Species-specific Prediction: We provide 185 species for species-specific prediction. If you want to focus on certain species, you may choose this one.
(5) GPS Web Server of Prediction by Protein Identifiers : If you want to predict with gene name, protein name or UniProt Accession, please choose this one.


4. Q: How to read the GPS 6.0 results?

A: Here we use the human protein ESR1 as the example. After clicking "Submit", the prediction results of MTOR-catalyzed sites with medium threshold are shown as follows:

<1>. The table of the GPS 6.0 results

ID: The name/id of the protein sequence that you input to predict.

Position: The position of the site which is predicted to be phosphorylated.

Code: The residue which is predicted to be phosphorylated.

Kinase: The regulatory kinase which is predicted to phosphorylate the site.

Peptide: The predicted phosphopeptide with 7 amino acids upstream and 7 amino acids downstream around the modified residue.

Score: The value calculated by GPS 6.0 algorithm to evaluate the potential of phosphorylation. The higher the value, the more potential the residue is phosphorylated.

Cutoff: The cutoff value under the threshold. Different threshold means different precision, sensitivity and specificity.

Source: Whether this phosphorylation site validated by experiment, "Exp." means YES, while "Pred." means NO. "Exp." links to the source site.

Links: The corresponding EPSD page.

Interaction: Whether the interaction between this substrate and this kinase validated by BioGrid, "√" that links to the source PubMed page, means YES, while "--" means NO.

Logo: The sequence logo of this phosphopeptide.

<2>. The visualization of default prediction

Part 1:
Up: The visualization for the positional distribution of the predicted site in protein sequence. By default, the sites with the highest 3 predicted scores are displayed.
Down: The visualization for protein disordered region predicted by IUPred [PMID: 15955779]. Cutoff = 0.5, if score of prediction > cutoff, the residue is considered in disordered region.

Part 2:
Left: The distribution of S/T/Y sites in kinase families.
     The distribution of S/T/Y sites.
     The distribution of S/T/Y sites in disordered region.

Right: The 3D structure of the substrate labeled with predicted phosphorylation sites.

<3>. The visualization of comprehensive prediction

Part 1:
Top: The surface accessibility of amino acids and the protein disordered region were predicted by NetSurfP ver. 1.1 (PMID: 19646261) and IUPred (PMID: 15955779), respectively. The cutoff of disordered region prediction = 0.5, if score of prediction > cutoff, the residue is considered in disordered region. The cutoff of surface accessibility prediction = 0.25, if score of prediction > cutoff, the residue is considered as surface exposed residue.
Bottom: The positions of the predicted phosphorylation sites were visualized in the protein sequence together with the secondary structure predicted by NetSurfP ver. 1.1 (PMID: 19646261).

Part 2 :
Left: The distribution of S/T/Y sites in kinase families.
Middle left: The distribution of S/T/Y sites.
Middle right: The distribution of S/T/Y sites in secondary structure.
Right: The distribution of S/T/Y sites in disordered region.

Part 3:
The 3D structure of the substrate labeled with predicted phosphorylation sites.


5. Q: How to choose the cut-off values and the thresholds?

A: Firstly, we calculated the theoretically maximal false positive rate (FPR) for each PK cluster. The three thresholds of GPS 6.0 were decided based on calculated FPRs.For serine/threonine kinases, the high, medium and low thresholds were established with FPRs of 2%, 6% and 10%. And for tyrosine kinases, the high, medium and low thresholds were selected with FPRs of 4%, 9% and 15%.


6. Q: What's the meaning of False Positive Rate (FPR)?

A: The false positive rate (FPR) is the proportion of negative sites that are erroneously predicted as positive hits. Given a data set containing all of non-phosphorylation sites, the real FPR could be easily computed. However, precise calculation of FPR is unavailable due to lack of a "gold-standard" negative data set. Here we developed a simple and fast method to construct the near-negative data set and estimate the theoretically maximal FPRs. Firstly, we calculated the distributions of amino acids composition in six organisms, including S. cerevisiae, S. pombe, C. elegans, D. melanogaster, M. musculus, and H. sapiens. Then we randomly generated 10,000 PSP(30,30) peptides to construct a near-negative data set based on the real frequencies of twenty amino acids in eukaryotic proteomes. Although there were a few sites to be real hits, its proportion would be very small. The process was repeated twenty times and the average FPR was calculated by GPS 6.0 as the theoretically maximal FPR. Also, the negative sites could be randomly retrieved from eukaryotic proteomes. And the results from both methods are very similar.


7. Q: How to use previous versions of GPS?

A: You can find the previous version of GPS at DOWNLOAD. Then download and install the GPS software that you need to your computer. GPS software is implemented in JAVA and could be installed on a computer with Windows/Linux/Unix/Mac OS. And we also wrote a manual for users which included in the installation package.


8. Q: I was trying to install the software on macbook pro but my installer says the file is damaged. How can I properly install the software in Mac OS?

A: By default, Mac OS 10.8 only allows users to install applications from 'verified sources'. In effect, this means that users are unable to install most applications downloaded from the internet. You can follow the directions below to prevent this error message from appearing.
(1) Open the Preferences. This can be done by either clicking on the System Preferences icon in the Dock or by going to Apple Menu > System Preferences.
(2) Open the Security & Privacy pane by clicking Security & Privacy.
(3) Make sure that the General section of the the Security & Privacy pane is selected. Click the icon labeled Click the lock to prevent further changes.
(4) Enter your username and password into the prompt that appears and click Unlock.
(5) Under the section labeled Allow applications downloaded from, select Anywhere. On the prompt that appears, click Allow From Anywhere.
(6) Exit System Preferences by clicking the red button in the upper left of the window. You should now be able to install applications downloaded from the internet.


9. Q: I have a few questions which are not listed above, how can I contact the authors of GPS 6.0?

A: Please contact the major author: Miaomiao Chen, Weixhi Zhang, and Dr. Yu Xue for details.


10. Q: Can I use GPS 6.0 on different browsers?

A: Yes, we tested our web server on different browsers.

Browser Compatibility
OSVersionChromeFirefoxMicrosoft EdgeSafari
LinuxUbuntu 18.04107.0.5304.107107.0.1N/AN/A
Windows10107.0.5304.107107.0.1112.0.1722.48 N/A