Overview

Introduction

The Overview tab is split into five main sections: the protein table at the top; the peptide and peptide to spectrum match (PSM) tables, both in the middle left; the spectrum view in the middle right; and the sequence coverage at the bottom.

All five sections are connected. This means that selecting a protein in the protein table updates the peptide table, the PSM table, the spectrum view and the sequence coverage. Similarly selecting a peptide in the peptide table updates the PSM table, the spectrum view and the sequence coverage etc etc.


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Proteins

PeptideShaker is designed for the robust identification of peptides and proteins in shotgun gel free experiments. The protein table lists all proteins that were found in the experiment.

For each protein, the protein inference status is color coded. If you click on the colored square, more details about the protein inference issue will be displayed.

The protein accession and protein description are given as parsed from the FASTA file. Note that we strongly encourage the use of UniProt databases. Clicking on the protein accession links then directly open the UniProt protein summary. The protein description is also given as parsed from the FASTA file. Once again, unrecognized databases may be result in issues or reduced amounts of information.

PeptideShaker estimates the protein coverage, number of peptides and spectra linking to this protein based on the validated peptides and PSMs. The validation of peptides and PSMs can be customized in the Validation tab.

Two protein protein quantification metrics are available in PeptideShaker:

You can select the desired spectrum quantification method in the Edit menu, Edit > Preferences.

The protein quantification methods are based on the validated identifications. It is important to note that the metrics are absolutely not precise and can be biased toward validation methods, normalization methods and shared peptides. They can however be normalized by known protein abundances and do give a rough estimate of the protein abundances.

The confidence in the protein identification is given in the last column. It important to stress here as well that the confidence should not be taken for granted. The confidence estimation is based on the Target/Decoy hits and thus suffers from accuracy limitations inherent in the method. Confidence estimation accuracy can be proofed in the Validation tab. If doubts in the confidence estimation accuracy remain, it is advised to verify the protein scores which can be accessed via the View menu.

The final column shows whether the protein has been validated or not, the validation criteria can be set in the Validation tab.


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Peptides

PeptideShaker displays in this table the peptides identified which can be linked to the currently selected protein.

For each peptide the Protein Inference Status is color coded. It can be that a peptide is shared between several proteins in the dataset and it is possible to inspect all cases by clicking on the given colored box.

The sequence of the peptide is displayed with the identified termini. The modifications are color coded using the colors set in the Search Parameters accessible in the Edit menu. For more information about post-translational modifications see the Modifications tab. The peptide's occurence(s) in the protein sequence are also displayed in the sequence coverage section at the bottom of the frame.

The number of validated PSMs supporting the peptide as well as the confidence and validation status are given in the three last columns. Like for proteins it is important to note that these are sensitive to the validation settings which can be fine tuned in the Validation tab.


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PSMs

The Peptide to Spectrum Matches (PSMs) supporting the selected peptide are displayed in this table. The peptide sequence with termini and colored modifications is displayed similarly as in the peptide table. Note that PSMs with different modification locations will link to the same peptide. The modification status of the peptide based on the PSMs can be optimized in the Modifications tab.

The SE column shows the search engine agreement, i.e., if the search eninges agree on the top ranking peptide or not. Click the colored box to inspect the search engine details.

The charge of the precursor as given by the search engine is given in this table. It is important to note that it might not correspond to the charge assigned by the mass spectrometer. The original charge can be found in the mgf file and inspected in the Spectrum IDs tab.

The difference in ppm between the measured precursor mass and the theoretic peptide mass is also provided. Note that PSMs with large precursor mass deviations are filtered out when loading the data.


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Spectrum

The spectrum corresponding to the selected PSM is located in the MGF file and dispayed by PeptideShaker. The spectrum annotation is independent of the search engine(s) and the corresponding settings can be fine tuned in the menu bar below the spectrum.

The annotation accuracy and the minimum annotation intensity level can be controlled by scrolling the mouse wheel (with and without holding doen the Ctrl key).

Depending on the peaks annotated, the peptide sequence coverage by the selected ion series is displayed with the corresponding ion intensities. An histogram displays the various peak intensities with annotated peaks in green and non-annotated peaks in gray. Finally, the mass error of the various peak annotations can be seen on the top right.

If one selects the Bubble Plot option the peaks will be represented by circles centered on the annotation mass error versus the fragment ion mass. The size of the bubbles represent the intensity of the ion. This display allows you to compare different PSMs. To do so, select multiple PSMs in the PSM table (by holding down the shift key).

The Ion Table shows which ions were found with the corresponding intensities. Note that it is here also possible to select different PSMs, the intensity variability will then be displayed with error bars. Note that fragment ion intensities of correct PSMs are usually highly reproducible.

For more help about the spectra display and annotation see the help accessible in the menu bar below the spectrum.


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Sequence Coverage

The sequence coverage is displayed for the selected protein. The validated peptides are displayed in green and the peptide selected in the peptide table is in blue. Areas if the sequence that is possible to cover with the given search settings is shown in light grey. PTMs are color coded and shown below the modified residue.

You can access a peptide by clicking on the corresponding peptide. In case of overlapping peptides you can select the peptide of interest using the appearing contextual menu.

It is also possible to zoom by left clicking an dragging the mouse towards the right.

Warning: the estimation of the possible sequence coverage is based on the distance between consecutive enzymatic cleavages only. It is a very simple approximation. Note that due to the presence of non-enzymatic cleavages and missed cleavages, search engines identify sections of the sequence that are not predicted to be coverable. Consequently, the observed sequence coverage might be higher than the possible sequence coverage.


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