Welcome to the PEAKS Peptide De Novo Sequencing tutorial. In this video I will outline the benefits of de novo sequencing and how it is a part of PEAKS. I will then show you how to perform a de novo sequencing analysis using PEAKS, which is the most widely accepted tool for peptide de novo sequencing in mass spec labs.
For peptide identification with tandem mass spec, de novo sequencing derives the peptide sequence without using a protein database. This can arise when researching unsequenced organisms, antibodies, endogenous peptides, and peptides with unexpected PTMs. Even when a sequence database is available, a database search engine can fail to assign database peptides to many high quality tandem mass spectra.
Researchers choose to use peaks because it is fast and most importantly, accurate. For example, in this third-party comparison of de novo sequencing algorithms, PEAKS outperformed all other algorithms compared in the paper.
Customer Testimonial (Outstanding User Interface)
PEAKS makes understanding de novo results easy, with an incredibly user-friendly interface. One publication in particular states:
“An important factor when performing large-scale de novo sequencing experiments is the ease of use and flexibility of the software. In this respect, PEAKS, being a commercial quality program, was far superior and offered the most adaptive interface, with the ability to import various formats of data from a vast array of mass spectrometers. The sequencing result displayed by PEAKS was also considerably more useful.”
Saves Time and Easy to Use
Using PEAKS is as simple as 1-2-3, as I will demonstrate for you now.
1. Select you data
2. Click the de novo sequencing icon
3. Specify your parameters, such as the error tolerance, enzyme, and PTM’s
PEAKS will then de novo sequence all of the tandem mass spectra in the data set, at a speed of up to 15 spectra per second on a regular PC, and even faster on a server.
Viewing De Novo Sequencing Results
Once the de novo sequencing process is finished, a de novo result node will appear below the selected data. It looks just like a small snow-capped mountain with “dn” in letters. Double click the node to open the result. The de novo sequences are listed in a table, along with the associated score and retention time for each sequence.
Selecting a peptide, will display the matching peptide-spectrum details, such as the spectrum annotation and the ion match table. You can easily zoom and navigate in the spectrum annotation using your mouse. This is an important feature if it is desired to examine individual peptides and as such there are several interesting ways to navigate the spectrum. Here are some examples:
1. drag to zoom
2. Use your scroll wheel to zoom into the y axis
3. Use your scroll wheel to zoom into the x-axis
Local Confidence Scores
To make things easy, individual amino acids are colour-coordinated, based on their respective confidence level. The confidence value can be examined by hovering your mouse over a particular peptide, red being above 90% is considered a great score, purple being between 80-90% is good, and blue representing 60-80% is acceptable. Anything below 60% is coloured black. This local confidence on each amino acid is a unique feature of PEAKS. It allows you to adjust the minimum local confidence threshold to convert the de novo sequence into a sequence tag that only contains the highly confident amino acids.
But what we really want to look at is the total local confidence (TLC) and average local confidence (ALC). The TLC score, indicates the expected number of correct amino acids, while the ALC score, indicates the expected percentage of correct amino acids. For example, here we have a TLC of 10.9 and an ALC of 84%, which indicates a confidently identified sequence.
While the default is to sort the table by TLC, you can sort by other columns by clicking the column title, or you can use the search function to quickly locate a peptide.
Summary View – Exporting Results
This summary view shows the result statistics. If you like you can filter the results by setting a score threshold. We recommend to start with the default TLC and ALC values, and adjust those values manually by examining the de novo sequences around this threshold.
The de novo sequencing results can be exported to text formats if you want to use PEAKS as a subroutine in your lab’s own workflow. To export the filtered results:
1. Click “Export” at the top of the summary view
2. Choose the format you wish to export the results in, available formats include html, csv, or xml format
3. Choose the location and directory name where you want to put the exported files
De Novo Sequencing is just the Beginning – PEAKS Workflow
For the analysis of mass spectrometry data, PEAKS does not stop at de novo sequencing. Instead, the de novo sequencing capability facilitates PEAKS to provide a number of unique benefits through several integrated tools. De novo sequencing is just the beginning.
First, the de novo sequencing results are used to confirm and improve PEAKS’ integrated database search. As a result, the performance of PEAKS is well above other database search software. For when sequences are not found by the database search engine, the peptides found exclusively by de novo sequencing can be analyzed by the integrated PTM finder and SPIDER tools, in order to locate unexpected PTMs and peptide mutations.
With PEAKS, you are not limited by only using a sequence database, which may be unavailable, incomplete, containing errors, or ineffective due to unexpected PTMs and mutations. PEAKS has all of the necessary tools to overcome these challenges, due to it’s unique de novo sequencing algorithm.
Thank you for watching our PEAKS de novo video. If you would like to learn more about how PEAKS can bring sensitivity and accuracy to your identifications, download a demo or check out more of our tutorial videos.