In a typical proteomics mass spectrometry analysis, a researcher needs to identify all peptides that produce good quality MS/MS spectra, whether or not the peptides are in a protein sequence database.
PEAKS is designed to facilitate such analyses. To enable accurate and sensitive peptide identifications, PEAKS provides an integrated tool set that features:
• de novo sequencing: to identify novel peptides
• database search: to identify database peptides
• SPIDER: to find peptides with PTMs and mutations
• inChorus: to increase coverage by combining multiple database search engines
• PEAKS Q module for protein quantification (optional)
In this short video, let’s focus on a typical result that has been prepared with the newly improved PEAKS DB workflow that combines de novo sequencing and database search to increase accuracy and sensitivity.
PEAKS DB & De Novo
The result consists of a result summary, the identified proteins, the identified database peptides, and a list of peptides identified exclusively by de novo sequencing. In the first tab of the results, PEAKS summarizes the results in an easy to read summary view. The summary view is the central place for result filtration and validation. The specification of a filter is as simple as clicking this FDR button, selecting the false discovery rate on this FDR curve, and clicking the apply filter button. The result statistics are then updated dynamically.
First, this bar graph shows the score distribution of the target and decoy hits. PEAKS uses an enhanced target decoy method to estimate the false discovery rate. Here we observe that there are very few decoy hits above the score threshold, indicating highly accurate results.
Second, this scatter-plot compares the precursor mass error with the peptide score. If you are using a high-resolution instrument, you should see that the error is small for the high-scoring peptides and starts to scatter for peptides below the score threshold.
To see the proteins inferred from the identified peptides, we simply select the protein tab located on the left. Take a look at the proteins discovered, along with the peptides associated to each protein and each proteins coverage sequences.
To take a closer look at the peptides confidently identified from the sequence database we select the peptide tab from the left navigation. Reported here are each peptides -10lgP score, annotated spectrum match, ion table, and error map.
Detailed Examination and Navigation
PEAKS also provides very user-friendly ways for you to examine an individual peptide that has been identified. Let’s say I want to check a peptide that contains an oxidized methionine, which has a mass shift of 15.99 daltons. Go to the peptide view, sort by the PTM type, and those peptides with the oxidized methionine will appear at the top of the list. Select an interesting peptide and the spectrum annotation will appear at the bottom. You can conveniently zoom and navigate the spectrum by using your mouse wheel, or, you may focus on a particular area by dragging the mouse and selecting a smaller area.
De Novo Only
The final tab labelled “De Novo Only” displays the results that are identified exclusively by de novo sequencing. Since these spectra do not match any significant peptides hits in the database, they are particularly interesting novel peptides that no other software can find.
As mentioned before, PEAKS studio is an integrated proteomics toolset. In addition to the renowned de novo sequencing algorithm, and the newly improved PEAKS DB module, PEAKS studio also consists of:
• SPIDER – A homology search tool that is designed to match your de novo results directly to the database. This allows you to identify peptides even when you are working with an unsequenced organism or highly variable proteins.
• inChorus – Which combines multiple database search engines’ results to increase the coverage, while comparing different engines’ performance side by side. InChorus is able to simultaneously run and compare: MASCOT, XTandem!, Sequest, OMSSA, and of course PEAKS.
• Quantification module (Optional)PEAKS Q – an optional quantification tool, that quantifies proteins using all common MS and MS/MS labelling methods. These methods can include: iTraq, ICAT, SILAC, Label Free, and other user-defined labels.
Thank you for watching our Overview video of PEAKS. 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.