Video Dialogue

Welcome to the PEAKS Database Search Tutorial on peptide identification. In this video, I will be going over the benefits and features of the newly improved PEAKS DB search engine, and demonstrating how to perform an analysis.

As this diagram illustrates, when you run PEAKS DB on raw MS/MS Spectra, the spectra are automatically de novo sequenced and the results are automatically combined with the database results. This gives you improved database results and can differentiate sequences exclusively identified by the de novo analysis. These exclusively de novo sequenced identifications can be potential novel peptides, or peptides with mutations and PTMs.

Now let’s take a closer look at PEAKS DB by performing a live demo of the workflow. Before you run PEAKS DB you need to make sure you have a database configured. It is only necessary to configure once. Step-by-step instructions on how to configure a database can be found in the PEAKS “Help” menu under “Help Contents”. The instructions are in Section I-Part 6 under “Configuring Sequence Databases“

With the new interface it is easy to run a PEAKS DB workflow:

1. Select your data
2. Select the PEAKS DB icon
3. Set the study parameters such as error tolerance, enzyme, fixed and variable PTM’s, as well as the desired database.
4. Once finished click OK.

One of the best features of PEAKS is the improved summary view. In this view you can easily filter and validate your results, as well as to get an overall understanding of the identifications.

To filter your results:

1. Select FDR from the toolbar
2. Navigate along the curve to find the desired percentage, we usually recommend using a 1% FDR
3. Click “Apply Filters”

In the Results Statistics section, users are able to visualize a graphical analysis of all peptides. The first figure summarizes the number of peptides spectrum matches, or PSMs, that are identified at the set FDR. Below, the figure on the left summarizes the number of identified PSMs and displays both the target and decoy identifications at each -10lgP score. The figure on the right shows the precursor error distribution. The error is small for high scoring peptides and scatters for those identified below the score threshold.

In the Experiment Control Section, the two figures can help check whether the instrument is well-calibrated. The left graph illustrates the distribution of the precursor mass error, where a distribution around 0 indicates a very well calibrated instrument. The right graph further plots the precursor error distribution against the precursor m/z

Once you have finished validating your results, it’s easy to export your results in a variety of formats. For example you can export to html, so that it may be integrated into a website. To do this:

1. Select Export
2. Choose html, additionally you can choose to export in csv, fasta, and xml formats
3. Choose the location
4. Click Export

In the Protein view we have a great view of each of the identified proteins. For each Protein it readily displays the associated -10lgP score, coverage percentage, number of peptides, number of peptides unique to that protein, and a brief description of the protein in the top pane. To take a closer look at a protein, select the protein and the associated peptides are displayed in the lower pane. You are also able to take a closer look at the protein’s coverage map by selecting the coverage tab in the lower pane.

In the peptide view we can see all of the identified peptides along with important details such as the -10lgp score, mass to charge ratio, and the Accession protein. Using the search box here, you can search a particular peptide by the scan number, peptide sequence and so forth. Or, you can sort the peptides using the column header, such as by PTM. This allows you to bring all peptides with a particular PTM to the top of the table. By selecting a peptide you are able to see its associated annotated spectrum, ion match table, and error map.

There are a couple of options available to refine the spectrum to display the information you desire. First there is an option to filter specific ions to display, found by selecting the wrench tool in the middle pane here. As a default, all b and y ions are selected, however to change this click the respective ions to add or remove from the spectrum. Once you have the desired ions displayed in the spectrum you can then zoom into an interesting area. To focus on a specific area click and drag your mouse, or you can use your mouse wheel to zoom into the x or y-axis. Double click to return to the original ratio.

The De Novo Only view displays all peptides that were not found in the database. To learn more about de novo sequencing results check out our PEAKS Peptide De Novo Sequencing Tutorial.

Thank you for watching the PEAKS DB tutorial, to see for yourself how powerful PEAKS DB is, download a free 30 day trial today. For more information, check out our website at bioinfor.com or send us an email to This e-mail address is being protected from spambots. You need JavaScript enabled to view it .