The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data (UniProt Consortium, 2023). The UniProt website receives about 800,000 unique visitors per month and is the primary means to access UniProt. Along with various datasets that you can search, UniProt provides four main tools. These are the “BLAST” tool for sequence similarity searching, the “Align” tool for multiple sequence alignment, the “Peptide Search” tool for retrieving proteins containing a short peptide sequence, and the “Retrieve/ID Mapping” tool for using a list of identifiers to retrieve UniProt Knowledgebase (UniProtKB) proteins and to convert database identifiers from UniProt to external databases or vice versa. This article provides four basic protocols and seven alternate protocols for using UniProt tools. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1 : Basic local alignment search tool (BLAST) in UniProt

Alternate Protocol 1 : BLAST through UniProt text search results pages

Alternate Protocol 2 : BLAST through UniProt basket

Basic Protocol 2 : Multiple sequence alignment in UniProt

Alternate Protocol 3 : Align tool through UniProt results pages and entry pages

Alternate Protocol 4 : Align tool through UniProt basket

Basic Protocol 3 : Peptide search in UniProt

Basic Protocol 4 : Batch retrieval and ID mapping in UniProt

Alternate Protocol 5 : Retrieve/ID Mapping tool through UniProt text search results pages and BLAST and Align results pages

Alternate Protocol 6 : Retrieve/ID Mapping tool through UniProt basket

Alternate Protocol 7 : Retrieve/ID Mapping tool through UniProt search box

UniProt, or the Universal Protein Resource, provides an up-to-date, comprehensive body of protein information at a single site (UniProt Consortium, 2023). The UniProt Knowledgebase (UniProtKB) delivers high-quality functional information for a selected set of protein sequences. To build upon these protein data and to aid analysis, UniProt provides four main tools: “BLAST” (Basic Local Alignment Search Tool), the “Align” multiple sequence alignment tool, “Peptide Search,” and “Retrieve/ID Mapping” for batch retrievals of UniProt entries and ID mapping between UniProt and external databases. These tools are available on their own dedicated pages on the UniProt website and are also accessible directly from other parts of the website, such as the basket, search/tool results pages, and protein entry pages. Having these tools in the UniProt website creates an integrated hub of data and analysis tools, allowing both to leverage each other. For example, if you come across sequences while browsing UniProt databases that you would like to BLAST or align, you can select your sequence and submit it to the relevant tool directly. Consequently, results from tools provide links directly to all relevant data from UniProt and allow you to filter by attributes, like whether you are looking for reviewed (UniProtKB/Swiss-Prot) or unreviewed (UniProtKB/TrEMBL) UniProtKB entries, entries with 3D structures, or entries that are part of a proteome, among others. All data in UniProtKB are freely available to users.

The UniProt website can be accessed at http://www.uniprot.org/. The following protocols describe how you can access and navigate the “BLAST” (Basic Protocol 1 and Alternate Protocols 1 and 2), “Align” (Basic Protocol 2 and Alternate Protocols 3 and 4), “Peptide Search” (Basic Protocol 3), and “Retrieve/ID Mapping” (Basic Protocol 4 and Alternate Protocols 5 to 7) tools on the UniProt website and use them in your analysis.

The UniProt website provides BLAST (UniProt Consortium, 2023), which finds regions of local similarity between sequences. This can be used to infer functional and evolutionary relationships between sequences as well as to help identify members of gene families. The BLAST tool page can be reached from a link in the header on all pages of the UniProt website.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Click on the “BLAST” link.

3.To run a BLAST search, enter either a UniProt identifier into the first input box or a protein or nucleotide sequence into the second input box provided.

4.Click on the “Run BLAST” button to execute the query.

As an alternative to Basic Protocol 1, queries can be submitted to the BLAST tool directly through UniProt search results pages and when you come across a sequence you would like to analyze using a sequence similarity search. This allows for a flexible workflow between browsing data and analyzing data.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Choose the search dataset using the drop-down menu to the left of the search box. Select “UniProtKB,” “UniRef,” or “UniParc.”

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.To run a BLAST search for a protein in the search results, click on the checkbox in the left-hand column for that protein row.

5.Click on the “BLAST” button just above the search results table, as shown in Figure 6, which will open the BLAST page, from where the BLAST can be launched as described in Basic Protocol 1, steps 3 and 4.

As an alternative to Basic Protocol 1, queries can be submitted to the BLAST tool directly through the UniProt basket feature. The UniProt basket allows you to store entries from UniProtKB, UniRef, or UniParc. You can use the basket to build a set of your proteins across different searches. The basket then allows you to download your dataset to access analysis tools (i.e., BLAST, Align, and Retrieve/ID Mapping). Your basket is saved as long as you do not clear your cookies.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Choose the search dataset using the drop-down menu to the left of the search box. Select “UniProtKB,” “UniRef,” or “UniParc.”

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.For the entry of interest, click on the checkbox to the left of its accession and then click on “Add” next to the basket icon button at the top of the results table, as shown in Figure 8.

5.When ready to analyze the entries in the basket, click on the basket to open it.

6.Click on the checkbox to the left of the entry of interest and then click on “BLAST,” as shown in Figure 9.

The UniProt website provides a multiple sequence alignment tool for proteins called “Align.” This tool runs the Clustal Omega algorithm to find areas of similarity in the entries being aligned. This can be used to find conserved residues and regions that can help infer evolutionary and functional relationships (see Current Protocols article: Simossis, Kleinjung, & Heringa, 2003).

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Click on the “Align” link.

3.To execute the multiple sequence alignment, enter either the UniProt identifiers into the first input box or the protein sequences in FASTA format into the second sequence input box provided and click “Align sequences.”

4.Once the alignment is finished, click on “completed” to open the Align results page, which is shown in Figure 13.

As an alternative to Basic Protocol 2, queries can be submitted to the Align tool directly through UniProt search results pages.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Select “UniProtKB” as the search dataset using the drop-down menu to the left of the search box.

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.Click on two or more checkboxes to align these protein entries, as shown in Figure 14.

5.Click on the “Align” button just above the search results table.

6.Click the “Align sequence” button.

7.Once the alignment is finished, click on “completed” to open the Align results page.

As an alternative to Basic Protocol 2, queries can also be submitted to the Align tool directly through the UniProt basket feature.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Select “UniProtKB” as the search dataset using the drop-down menu to the left of the search box.

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.For entries of interest, click on the checkboxes to the left of their accession numbers in the results table and then click on “Add” next to the basket icon at the top of the results table.

5.When ready to analyze the entries in the basket, click on the basket to open it, as shown in Figure 15.

6.Click on the checkboxes to the left of the entries to be aligned and then click on “Align.”

7.Click the "Align sequence" button.

8.Once the alignment is finished, click on “completed” to open the Align results page.

The UniProt website provides a tool that allows you to upload short peptide sequences of at least three residues and find all UniProtKB sequences that have an exact match to the query sequence. These peptide sequences can come from proteomics experiments or from the design of peptides for antibody production, for example.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Click on the “Peptide search” link.

3.Enter the peptide(s) of interest into the input box as shown in Figure 17 and click on the “Run Peptide search” button.

4.Once the search is finished, click on “completed” to open the results page, which is shown in Figure 19.

The UniProt website provides a tool that allows you to upload a list of UniProt identifiers and batch-retrieve all the corresponding UniProt entries. It allows you to convert or “map” your identifiers from UniProtKB to over 100 external databases that UniProt is cross-referenced to and vice versa (e.g., Ensembl, PDB, RefSeq; Huang et al., 2011). This covers a number of databases from different categories, including databases covering sequence, 3D structure, protein-protein interaction, protein family and group, chemistry, post-translational modification, and genome annotation, among others. This tool is called “Retrieve/ID Mapping.”

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Click on the “ID Mapping” link.

3.To retrieve functional information or look at the sequences for a list of UniProt IDs (for example, Q9VR99, Q9VEZ5, Q03017, and P48607), paste the list into the input box provided or upload a file.

4.Leave “From” and “To” as “UniProtKB” and click the “Map IDs” button.

5.Once the mapping is finished, click on “completed” to open the results page as shown in Figure 22.

6.To map a list of UniProt IDs to IDs from an external database such as Ensembl, RefSeq, or one of the model organism databases (or vice versa), upload or paste in the IDs. Then, select the source database in the “From” drop-down menu and the target database in the “To” drop-down menu.

7.Click the “Map IDs” button as shown in Figure 23.

As an alternative to Basic Protocol 4, queries can be submitted to the “Retrieve/ID Mapping” tool directly through UniProt search results pages, BLAST results pages, or Align results pages.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Select “UniProtKB” as the search dataset using the drop-down menu to the left of the search box.

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.Click on two or more checkboxes as shown in Figure 24.

5.Click on the “MapIDs” button just above the search results table.

Queries can be submitted to the “Retrieve/ID Mapping” tool directly through the UniProt basket. The UniProt basket allows you to store entries from UniProtKB, UniRef, or UniParc. You can use the basket to build a set of your proteins across different searches. The basket then allows you to download your dataset to access analysis tools (i.e., BLAST, Align, and Retrieve/ID Mapping). Your basket is saved as long as you do not clear your cookies.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Choose “UniProtKB” for the search dataset using the drop-down menu to the left of the search box.

3.Enter a query in the search box, for example “insulin,” and click on the “Search” button.

4.For entries of interest, click on the checkboxes to the left of their accession numbers in the results table and then click on “Add” next to the basket icon at the top of the results table, as shown in Figure 8.

5.When ready to analyze the entries in the basket, click the basket to open it.

6.To map the UniProt IDs to an external database from the basket, click on the checkboxes to the left and click on the “Map IDs” button in the basket, as shown in Figure 25.

As an alternative to Basic Protocol 4, queries can be submitted to the “Retrieve/ID Mapping” tool directly through the UniProt search box.

Necessary Resources

• An up-to-date web browser

1.Go the UniProt home page at http://www.uniprot.org/ using an up-to-date web browser.

2.Click on “List” on the left-hand side of the general search box as shown in Figure 26.

UniProt BLAST Results

The UniProt BLAST results appear as shown in Figure 4. The results page provides (1) a left-hand side panel with filters and (2) a results table that contains the protein entries (hits) that match your query. The left-hand side panel allows you to filter the results based on the percent identity, the score, or the E-value. You can also filter for reviewed entries (UniProtKB/Swiss-Prot), unreviewed entries (UniProtKB/TrEMBL), entries with specific features such as proteins with a 3D structure, or entries from a specific organism. For example, Figure 27 shows how you can use the filter by taxonomy to display only mouse entries.

In the results table, the protein entries are sorted according to their score in descending order. For each entry, various information is displayed, including the name, the organism, and the protein length. The last column on the left provides a basic graphical view of the sequence alignment showing the percent identity, the score, and the E-value in small boxes. Clicking on the sequence box opens a window at the bottom of the page where you can see a detailed view of the alignment between the query and the matched entry, as shown in Figure 28. The alignment can be explored as described in the UniProt Align Results section below.

On the top of the page, various tabs allow you to view the results according to their “Taxonomy,” or “Hit Distribution,” or as a “Text Output.”

You can download your alignment in various formats by clicking on the “Download” button. You can also select one or multiple entries by clicking the checkboxes next to them to run a BLAST or an alignment or to store the entries in the basket for further analysis. Using the “Customize columns” button above the table allows you to change the type of information displayed. For example, in Figure 29, the subcellular location is shown.

The “API request” tab provides you with the code to run the current job with the same input on the command line using curl.

You can save the URL of your sequence alignment to access it at any time for up to 7 days from when you first ran the query. The “Input Parameters” tab provides detailed information about the alignment parameters, including the job identifier, which can be used to access your sequence alignment at any time for up to 7 days from when you first ran the query.

UniProt Align Results

Multiple sequence alignments can help understand evolutionary conservation of structurally and functionally important regions of protein sequences (see Current Protocols article: Simossis et al., 2003). To obtain meaningful results and minimize errors in the alignment, it is necessary to align sequences that are likely to be related to each other.

The UniProt Align results appear as shown in Figure 13. The results page displays the full sequence alignment by default (“wrapped” view). You can change how the alignment is displayed by selecting one of the “view” options above the sequence on the right. The “overview” mode allows you to zoom in using the gray sequence toggles and explore a specific region of the alignment, as shown in Figure 30.

Above the aligned sequence on the left, there are two drop-down menus that allow you to select sequence annotations (i.e., domains, sites) to view them highlighted across the aligned sequences. For example, in Figure 31, the active site has been selected using “Select annotation” and is shown highlighted in the sequence. Clicking on the active-site pictogram opens a box where you can find additional information about the site. You can also highlight sequence features by amino acid properties (i.e., hydrophobicity). Both menus are available in the “overview” and “wrapped” views.

On the top of the results page, there are various tabs that give you the option to change how the alignment is visualized. The alignment can be viewed as “Trees,” as shown in Figure 32; as “Percent Identity Matrix;” or as “Text Output.”

You can download your alignment in various formats by clicking on the “Download” button. You can also select one or multiple entries by clicking the checkbox next to them to run a BLAST or a new alignment or to store the entries in the basket for further analysis.

The “API request” tab provides you with the code to run the current job with the same input on the command line using curl.

You can save the URL of your sequence alignment to access it at any time for up to 7 days from when you first ran the query. The “Input Parameters” tab provides detailed information about the alignment parameters, including the job identifier, which can be used to access your sequence alignment at any time for up to 7 days from when you first ran the query.

UniProt Peptide Search Results

The “Peptide Search” results provide UniProtKB entries with sequences that contain an exact match for each peptide that you searched for.

The results page for the peptide search is shown in Figure 19. For each protein entry row, the position of the peptide in the sequence and the sequence of the peptide are provided in the “Match” column. The filters on the left-hand side can be used to restrict the search to specific features. For example, you can display only UniProtKB/Swiss-Prot/reviewed entries.

Protein entries can be selected by clicking the checkboxes and then can be downloaded or stored in the basket for analysis at a later time point.

The “API request” tab at the top of the results page provides you with the code to run the current job with the same input on the command line using curl.

You can also save the URL of your peptide search to access it at any time for up to 7 days from when you first ran the query. The “Input Parameters” tab provides detailed information about the parameters, including the job identifier, which can be used to access your peptide results table at any time for up to 7 days from when you first ran the query.

UniProt Retrieve/ID Mapping Results

If you retrieve a batch of UniProt entries for a list of IDs using this tool, you will get a results page as shown in Figure 22. This results page provides filters in the left-hand side panel and a main results table. The results table starts with a column titled “From” that shows your input identifiers. The next columns in the results table show information from the corresponding UniProt entries that were found. You can edit these columns by clicking on the “Customize Columns” button above the results table. You can also run tools such BLAST and Align and add entries to your basket by selecting the corresponding checkboxes and then clicking on the buttons available at the top of the results page. You can download the full results table or just the list of identifiers using the “Download” button.

If you use the tool to map UniProt IDs to external database IDs (or vice versa; Huang et al., 2011), you will get a results table with two columns showing your input IDs and the corresponding mapped IDs, as shown in Figure 23. You can use the “Download” button to download your results.

The “API request” tab provides you with the code to run the current job with the same input on the command line using curl.

You can save the URL of your ID mapping to access it at any time for up to 7 days from when you first ran the query. The “Input Parameters” tab provides detailed information about the parameters, including the job identifier, which can be used to access your mapping results table at any time for up to 7 days from when you first ran the query.

Background Information

UniProt aids scientific discovery by collecting, interpreting, and organizing information so that it is easy to access and use. In addition to providing data through various datasets, UniProt also provides tools to help researchers analyze these data. UniProtKB is the central hub for the collection of functional information and other rich annotations on proteins. It is further divided into the Reviewed (UniProtKB/Swiss-Prot), expertly annotated section and the Unreviewed (UniProtKB/TrEMBL), automatically annotated section. UniParc is a non-redundant archive containing all the publicly available protein sequences in the world. UniRef provides clustered sets of sequences from UniProtKB (including isoforms) and selected UniParc entries. UniRef reduces redundancy and provides complete coverage of the sequence space at three levels of sequence identity (i.e., 100%, 90%, and 50% identity). The Proteomes dataset provides protein sets for organisms with completely sequenced genomes. Supporting datasets are a collection of meta-information about proteins in UniProtKB entries, such as literature citations, taxonomy, subcellular locations, keywords, cross-referenced databases, and diseases. The tools UniProt provides are BLAST, Align, Peptide Search, and Retrieve/ID Mapping. The UniProt website was designed following a user-centered design process and is flexible, powerful, and user friendly. It provides many ways of accessing these tools. Using tools within UniProt, you can easily chain activities by (1) searching for data, (2) running a BLAST search for a sequence in your results, (3) running a multiple sequence alignment on sequences in your BLAST results, and then (4) mapping the IDs of these sequences to an external database.

UniProt provides training material through the European Bioinformatics Institute (EMBL-EBI) online training portal, including a quick tour (https://www.ebi.ac.uk/training/online/courses/uniprot-quick-tour/) and a detailed course (https://www.ebi.ac.uk/training/online/courses/uniprot-exploring-protein-sequence-and-functional-info/). UniProt also provides short video tutorials embedded in the website, and they are available on a YouTube channel at https://www.youtube.com/uniprotvideos.

Critical Parameters

The Tools dashboard is where all the results of the Tools queries that you have run are stored for 7 days from when you first ran the query. You can access the dashboard by clicking the toolbox icon on the right-hand side of the UniProt head banner as shown in Figure 33. For each job, you have the option to resubmit the query, with the possibility to modify the parameters, store the job for more than 7 days (this requires the user to not clear UniProt website cookies), or delete the job.

Acknowledgments

This work was supported by the National Human Genome Research Institute (NHGRI), Office of the Director (OD/DPCPSI/ODSS), National Institute of Allergy and Infectious Diseases (NIAID), National Institute on Aging (NIA), National Institute of General Medical Sciences (NIGMS), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Eye Institute (NEI); National Cancer Institute (NCI), and National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health under Award Number [U24HG007822].

Open access funding enabled and organized by Projekt DEAL.

Author Contributions

Rossana Zaru : Writing – original draft, writing – review and editing; Sandra Orchard : Funding acquisition, writing – review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data in UniProt are freely available and can be accessed at https://www.uniprot.org.

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